master thesis work 2010 - Sociedade Brasileira de Fruticultura

Transcrição

Agricultural Science and Resource Management in the
Tropics and Subtropics (ARTS)
Department of Phytopathology & Nematology in Soil Ecosystems (INRES)
Occurrence, incidence and grower perception of
Fusarium oxysporum f.sp. cubense in banana intercropped with
coffee and trees in Costa Rica and Nicaragua smallholders.
Thesis
In partial fulfilment of
the requirements for the
academic degree of
Master of Science
of the
Faculty of Agriculture
Rheinische Friedrich-Wilhelms-Universität Bonn
Submitted on 20th May 2010
by
Paulo dos Santos Faria Lichtemberg
Brazil
Department of Phytopathology & Nematology in Soil Ecosystems (INRES)
Occurrence, incidence and grower perception of
Fusarium oxysporum f.sp. cubense in banana intercropped with
coffee and trees in Costa Rica and Nicaragua smallholders.
Thesis
In partial fulfilment of
the requirements for the
academic degree of
Master of Science
of the
Faculty of Agriculture
Rheinische Friedrich-Wilhelms-Universität Bonn
Submitted on 20th May 2010
by
Paulo dos Santos Faria Lichtemberg
Brazil
Main supervisor
Prof. Dr. Richard Sikora
Co-supervisor
Dr. Alexander Schouten
Chair person
Prof. Dr. Holger Hindorf
Acknowledgments
Dr. Professor Richard A Sikora and Dr. Luis E. Pocasangre,
For the opportunity and trust.
Dr. Charles Staver, Dr. Oscar Bustamante,
For the intense work.
Cindy Castillo, Ligia Quezada, David Brown, Juan Ortiz, Manrique González and Nancy
Chaves,
For the friendship and field work support.
M.Sc. Christian Dold,
For the patience friendship and the experience we share during our field arbeit.
Dr. Alfonso J. Cabrera and Dr. Roy Menjivar,
For your intense friendship and advices.
M.Sc. Professor Wilber Salazar, David Concepción and M.Sc. Juan Castillón,
For the assistance in Nicaragua.
The farmers,
For the interest and receptivity.
Profes. Richard M. Miller, Jucinei J. Comin, Antonio Uberti and Moisés Soto Ballestero,
For the guidance at the beginning of my academic career.
Rafael Schadeck, Rodrigo Morigutti, Andres Mendonça, Patricia Barroso, Priscila
Brandão, Maria Arias, Dominika Schneider and Pangestuti Astri,
There is no life without friends.
Dedication
Vera and João, Luiz and Beatriz;
Vó Elia and Vó Lindaura;
Juliana, Fábio and Helmut;
Baco and Jonas.
I love you all so much.
Paulo dos Santos Faria Lichtemberg M.S.c. Thesis
Table of Contents
Table of Contents..................................................................................................................................... i
List of Abbreviations .............................................................................................................................. iv
List of Tables ........................................................................................................................................... v
List of Figures ......................................................................................................................................... vi
Summary ............................................................................................................................................. viii
1.
General Introduction ....................................................................................................................... 1
1.1.
Bananas ................................................................................................................................... 1
1.2.
Fusarium Wilt........................................................................................................................... 3
1.2.1.
The Pathogen ................................................................................................................... 3
1.2.2.
Physiologic Races and Vegetative Compatibility Groups .................................................... 4
1.2.3.
Fusarium oxysporum f. sp. cubense Biology ...................................................................... 5
1.2.4.
Infection and Dissemination ............................................................................................. 6
1.2.5.
Fusarium Wilt Symptoms .................................................................................................. 6
1.2.6.
Fusarium Wilt Occurrence in Central America ................................................................... 8
1.2.7.
Control Methods............................................................................................................... 8
1.3.
2.
Objectives .............................................................................................................................. 10
Fusarium Wilt Incidence and Severity ............................................................................................ 11
2.1.
Introduction ........................................................................................................................... 11
2.2.
Experimental Site ................................................................................................................... 11
2.3.
Material and Methods............................................................................................................ 12
2.3.1.
Farm Selection ................................................................................................................ 12
2.3.2.
Plot Setting ..................................................................................................................... 12
2.3.3.
Base Line Study............................................................................................................... 13
2.3.4.
Detailed Study ................................................................................................................ 13
i
3.
4.
2.4.
Statistics ................................................................................................................................ 15
2.5.
Results ................................................................................................................................... 15
2.5.1.
Farms Profile .................................................................................................................. 15
2.5.2.
Costa Rica....................................................................................................................... 19
2.5.3.
Nicaragua....................................................................................................................... 22
2.6.
Discussion .............................................................................................................................. 25
2.7.
Conclusion ............................................................................................................................. 28
2.8.
Recommendation................................................................................................................... 28
The Fusarium Wilt Traditional Knowledge. ..................................................................................... 29
3.1.
Introduction ........................................................................................................................... 29
3.2.
3.3.
Materials and Methods .......................................................................................................... 30
3.4.
Statistics ................................................................................................................................ 30
3.5.
Results ................................................................................................................................... 31
3.5.1.
Banana Importance ........................................................................................................ 31
3.5.2.
Banana Management ..................................................................................................... 32
3.5.3.
Fusarium Wilt (Foc) Traditional Knowledge ..................................................................... 33
3.5.4.
Seed Management ......................................................................................................... 37
3.6.
Discussion .............................................................................................................................. 38
3.7.
Conclusion ............................................................................................................................. 40
3.8.
Recommendation................................................................................................................... 41
Investigation on the Pathogenicity of CR Isolates of Foc. ................................................................ 42
4.1.
Introduction ........................................................................................................................... 42
4.2.
4.3.
Material and Methods............................................................................................................ 43
ii
4.3.1.
Plant Material ................................................................................................................ 43
4.3.2.
Isolate Preparation ......................................................................................................... 43
4.3.3.
Plant Inoculation ............................................................................................................ 44
4.3.4.
Media Preparation ......................................................................................................... 45
4.3.5.
Treatment Application .................................................................................................... 45
4.4.
Nicaraguan Isolates ................................................................................................................ 46
4.5.
Statistics ................................................................................................................................ 46
4.6.
Results ................................................................................................................................... 47
4.6.1.
Morphological Characterization...................................................................................... 47
4.6.2.
Field Sampling and Vascular Tissue Colonization ............................................................. 49
4.6.3.
Pathogenicity Test – Incidence and Severity .................................................................... 51
4.6.4.
Pathogenicity Test – Growth Variables ........................................................................... 53
4.6.5.
Categories Analysis ......................................................................................................... 53
4.6.6.
Segment Colonization ..................................................................................................... 56
4.7.
Discussion .............................................................................................................................. 56
4.8.
Conclusion ............................................................................................................................. 58
4.9
Recommendation................................................................................................................... 58
5.
References..................................................................................................................................... 59
6.
Annexes ......................................................................................................................................... 69
7.
6.1.
Annex 1 – Field Form (FF001) ................................................................................................. 69
6.2.
Annex 2 – Questionnaire ........................................................................................................ 70
6.3.
Annex 3 – Fusarium Culture Identification (FCI003) ................................................................ 74
Curriculum Vitae ............................................................................................................................ 75
iii
List of Abbreviations
APOT:
Asociación de los Productores Orgánicos de Turrialba (Turrialba organic
farmers asociation).
BLS:
Base Line Study.
CATIE:
Centro Agronómico Tropical de investigación y Enseñanza (Tropical
Agricultural Research and Higher Education Center).
CR:
Costa Rica (correspond to zone 1 and 2 in Costa Rica study area).
DS:
Detailed Study.
Foc:
Fusarium oxysporum f. sp. cubense.
INIBAP:
International Network for Improvement of Banana and Plantain.
NIC:
Nicaragua (correspond to zone 1 and 2 in Nicaragua study area).
OIRSA:
Organización Internacional Regional de Sanidad Agropecuaria (Regional
International Organization of Animal and Agricultural Sanity).
PDA:
Potato Dextrose Agar.
UCR:
Universidad de Costa Rica (University of Costa Rica).
UNAN:
Universidad Nacional Autónoma de Nicaragua (Nicaraguan Autonomy
National University).
VCG:
Vegetative Compatibility Group.
iv
List of Tables
Table 1: Grouping of Fusarium oxysporum f. sp. cubense according VCG, race and
geographical distribution. ..................................................................................................................... 5
Table 2: Scale for rating external symptoms caused by Fusarium oxysporum f. sp. cubense. ... 14
Table 3: Statistical correlation of Fusarium Wilt incidence and field data measures in Costa
Rica. ..................................................................................................................................................... 21
Table 4: Statistical correlation of Fusarium Wilt incidence and field data measures in Nicaragua.
.............................................................................................................................................................. 24
Table 5: Statistical analysis of Fusarium Wilt incidence among countries .................................... 25
Table 6: Morphological characteristics of Fusarium isolates recovered from Costa Rican
diseased banana. ............................................................................................................................... 49
Table 7: Morphological characteristics of Fusarium isolates recovered from Nicaraguan
diseased banana. ............................................................................................................................... 49
Table 8: Fusarium spp. isolates origin by vascular tissue type ...................................................... 51
Table 9: Incidence and severity of 17 Fusarium oxysporum f. sp. cubense isolates in ‘Gros
Michel’ banana after 6 weeks ............................................................................................................ 52
Table 10: Effect of 17 Fusarium oxysporum f. sp. cubense isolates on plant growth of Gros
Michel in-vitro plants over 6 weeks. .................................................................................................. 53
Table 11: Categories of virulence, external and internal symptoms of Fusarium Wilt.................. 54
Table 12: Categories of virulence, growth development. ................................................................ 54
Table 13: Statistical correlation between incidence, severity and growth variables of
pathogenicity test treatments............................................................................................................. 56
v
List of Figures
Figure 1: Diagrammatic representation of a fruiting banana plant with suckers .......................... 2
Figure 2: External and internal symptoms of Fusarium Wilt ........................................................ 7
Figure 3: Experimental Sites of Costa Rica and Nicaragua .......................................................12
Figure 4: Plot setting stages ......................................................................................................13
Figure 5: Scale of rating internal symptom caused by Fusarium Wilt.........................................14
Figure 6: Vascular tissue sampling locations.............................................................................15
Figure 7: Farm and associated banana/coffee size in Costa Rica & Nicaragua smallholders ....16
Figure 8: Land usage in Costa Rica & Nicaragua smallholders .................................................17
Figure 9: Altitude and Inclination of Costa Rica & Nicaragua smallholders ................................17
Figure 10: Tree, coffee and banana participation in Costa Rica & Nicaragua smallholders .......18
Figure 11: Banana type participation in Costa Rica & Nicaragua smallholders ..........................18
Figure 12: Banana and coffee production in Costa Rica & Nicaragua smallholders ..................19
Figure 13: Occurrence of Fusarium Wilt in Costa Rica smallholders .........................................20
Figure 14: Farm localization map and Fusarium Wilt occurrence in Costa Rica. .......................20
Figure 15: Fusarium Wilt plant incidence in Costa Rica and correspondent zones ....................21
Figure 16: External and internal severity of Fusarium Wilt in Costa Rica smallholders ..............22
Figure 17: Occurrence of Fusarium Wilt in Nicaragua smallholders ..........................................23
Figure 18: Farm localization map and Fusarium Wilt occurrence in Nicaragua .........................23
Figure 19: Fusarium Wilt plant incidence in Nicaragua and correspondent zones. ....................24
Figure 20: External and internal severity of Fusarium Wilt in Nicaragua smallholders ...............25
Figure 21: Interview results about ‘crop importance’ in Costa Rica & Nicaragua .......................31
Figure 22: Interview results about ‘fertilization management’ in Costa Rica & Nicaragua ..........32
Figure 23: Interview results about ‘banana management and training’ in Costa Rica & Nicaragua
.................................................................................................................................................33
Figure 24: Interview results about ‘Fusarium Wilt knowledge’ in Costa Rica & Nicaragua .........34
Figure 25: Interview results about ‘Fusarium Wilt diagnoses’ in Costa Rica & Nicaragua ..........35
Figure 26: Interview results about ‘Fusarium Wilt dissemination and control’ in Costa Rica &
Nicaragua .................................................................................................................................36
Figure 27: Nicaragua Correspondence Analyses: traditional knowledge and incidence ............37
Figure 28: Interview results about ‘seed management’ in Costa Rica & Nicaragua ...................38
Figure 29: Isolate preparation steps. .........................................................................................43
Figure 30: Pathogenicity preparation steps ...............................................................................43
vi
Figure 31: Color variation of Fusarium isolates recovered from Gros Michel in Costa Rica. ......48
Figure 32: Color variation of Fusarium isolates recovered from Gros Michel in Nicaragua. .......48
Figure 33: Fusarium colonization levels in Costa Rica & Nicaragua banana .............................50
Figure 34: Effect of 17 Fusarium oxysporum f. sp. cubense isolates in Gros Michel banana .....52
Figure 35: Category 1 of incidence, severity and growth ...........................................................55
Figure 36: Category 4 of incidence, severity and growth ...........................................................55
vii
Summary
Occurrence, incidence and grower perception of Fusarium oxysporum f.sp.
cubense in banana intercropped with coffee and trees in Costa Rica and
Nicaragua smallholders.
Fusarium oxysporum f. sp. cubense, the causal agent of Fusarium Wilt is an
important disease for the growers of ‘Gros Michel’ dessert bananas throughout the
world. ‘Gros Michel’ bananas in central Costa Rica and northern Nicaragua are grown
primarily intercropped with coffee and trees, for home consumption and the national
market. Little research has been done on ‘Gros Michel’ in this smallholder production
system, where the problems with Fusarium Wilt potentially could occur. In this study the
occurrence and incidence of Fusarium Wilt was evaluated and grower knowledge and
management of the disease were characterized. The Fusarium Wilt survey was
performed on 30 farms of central Costa Rica and northern Nicaragua showing
occurrences of 33% and 23% respectively. The diseased plant incidence in central
Costa Rica was 7.31% and while in northern Nicaragua 1.12%. Significant difference
among countries incidence was not revealed, however an important difference were
noted among northern Nicaraguan zones, representing a good opportunity for diseasefree banana seed production. The traditional grower knowledge about banana
production and Fusarium Wilt control method were characterized, and results
demonstrate that the lack of training may be related to the disease incidence and would
be a threat for the disease dispersion. In northern Nicaragua 18 isolates were recovered
from diseased banana plants, been all morphologically identified as Fusarium spp. while
in central Costa Rica 17 isolates were recovered by diseased plants and through a
greenhouse bioassay were identified as Fusarium oxysporum f. sp. cubense, with three
of them offering lower virulence and demonstrating potential antagonist effect as biocontroller.
viii
Chapter 1 General Introduction
CHAPTER 1
1. General Introduction
The research work presented here concerns a survey of the occurrence and
incidence of Fusarium Wilt disease in Musa spp. affecting smallholders of the Turrialba
region, in Costa Rica, and the San Rámon and Jinotega regions in Nicaragua. For
clarity the results of this survey and associated research program is divided into three
sections: 1) methodology used to determine the incidence and severity of Fusarium Wilt
2) techniques used to evaluate traditional knowledge concerning banana management
and disease control, and 3) details of laboratory and bioassays performed with
Fusarium Wilt on banana to complement the information generated in the survey. On
this document the studied zones of Costa Rica and Nicaragua will be referred as CR
and NIC.
1.1.
Bananas
The Banana was first described in the eighteenth century by the Swedish
botanist, physician and zoologist Carl Linnaeus, who gave its name in 1750, simply
adapted from Arabic for banana, “mauz” (Koeppel 2008). Linnaeus also translates the
name of the yellow and sweet banana as Musa sapientum from the Latin term meaning
“wise” (Cordeiro et al. 2004, Koeppel 2008). Far from their center of origin the Musa
species became one of the most important cultivated crops. Worldwide more than 120
countries are involved in the production of bananas (Cordeiro et al. 2004). In tropical
areas it is one of the most important staple foods for more than 400 million people
(IPGRI 2000). Local market banana trade is one of the few activities that provide
households regular income throughout the year (Arias 2003).
Bananas are monocotyledons of the genus Musa, family Musaceae. The genus
Musa embraces four sections, Australimusa, Callimusa, Rhodochlamys and Eumusa
(Wardlaw 1961, Stover and Simmonds 1987, Daniells et al. 2001, Soto 2008). The
majority of cultivated and edible bananas arose from the Eumusa section, being the
biggest and geographically most ranging section of the genus (Stover and Simmonds
1987, Daniells et al. 2001). Most of cultivated bananas are triploid, and had their origin
1
in two wild, diploid, and fertile species, M. acuminate and M. balbusiana (Wardlaw 1961,
Stover and Simmonds 1987, Jones 2000). From these two species the banana
classification code was developed, with the number of As and Bs, designating the
genomic composition and ploidity level (Stover and Simmonds 1987).
Banana is a large, herbaceous plant consisting of branched, underground stems,
also called a corm or rhizome, that have roots and vegetative buds from where arise an
erect pseudostem composed by tightly packed leaf bases (Jones 2000). At a certain
point of development the oldest pseudostem, known as mother plant, stops producing
leaves and develops the inflorescence and fruits (Jones 2000). The lateral offshoots are
called suckers. The complex of mother and sucker plants, including roots and
pseudostems is called a banana mat (Stover and Simmonds 1987). The diagrammatic
representation of a banana mat can be seen in Figure 1.
Figure 1: Diagrammatic representation of a fruiting banana plant with suckers (Source: Jones, 2000).
2
There is some evidence that banana plants may have been introduced through
Santo Domingo by Fray Thomás de Berlanga, Bishop of Panamá, from the Canary
Islands in 1516. It was then disseminated to neighbor islands until it reach the continent
(Soto 2008). In Costa Rica, banana cultivation started in 1872 with seeds brought from
Panamá. Seven years later the export industry began operation with the Tropical
Trading & Transport Company (Soto 2008). Long the Atlantic zone of Nicaragua, in
1899 Bluefields Steamship (United Fruit Co) started banana production for export (Soto
2008). Today, Costa Rica and Nicaragua is the 2nd and 11th biggest banana exporting
countries in Latin America (FAO 2006). In 2007 banana production area in Costa Rica
corresponded to 43.000 ha with production of 2.240.000 tons while Nicaragua
corresponded to 879 ha producing 47.072 tons (OIRSA 2008). In contrast to the export
industry, the internal market in Latin America and The Caribbean absorb 50% of the
produced banana crop (Pocasangre and Pérez-Vicente 2010). The ‘Gros Michel’ (AAA)
cultivar is now only produced in Costa Rica and Nicaragua by smallholders usually
intercropped with coffee due to the Fusarium Wilt constraint (Lara et al 2009).
1.2.
Fusarium Wilt
1.2.1. The Pathogen
Fusarium Wilt, caused by the soilborne pathogen Fusarium oxysporum f. sp.
cubense, here referred as Foc, is one of the most destructive plant diseases recorded in
history (Wardlaw, 1961, Ploetz 1994, Moore et al. 1995, Ploetz and Pegg 2000,
Pocasangre 2009, Orozco et al. 2009, Pérez-Vicente and Pocasangre 2010). It was first
described in Australia by Bancroft in 1876 (Moore et al. 1995, Ploetz 2005b, PérezVicente and Pocasangre 2010) but became most important in the monocultures of ‘Gros
Michel’ (AAA) where Foc destroyed from around 40.000 ha of banana plantations
between 1890 to 1960 in Central and South America (Stover and Simmonds 1987,
Ploetz 1994, Ploetz and Pegg 2000). Reports at that time, indicated losses of 22.000 ha
in Panamá, 13.000 ha in Costa Rica, 3.000 ha in Honduras and 2.200 ha in Guatemala
(Wardlaw 1961). ‘Gros Michel’ (AAA) export trade suffered enormous economic losses
and was consequently forced to convert to cultivars of ‘Cavendish’ (AAA) subgroup
3
(Ploetz 1994, Ploetz and Pegg 2000, Molina 2009, Pocasangre and Pérez-Vicente
2009).
1.2.2. Physiologic Races and Vegetative Compatibility Groups
Fusarium oxysporum is a very complex species, composed of around 70 strains
with specific hosts and a huge number of non-pathogenic saprophytes (Pérez-Vicente
2010, Pérez-Vicente and Battle 2010a). In Foc there is great strain variation and several
methods have been developed to separate them (Davis 2005). Strains have most
commonly been grouped by their ability to cause disease in different banana types in
the field. They have been described as ‘races’ 1, 2, 3 or 4. Race 1 was responsible for
the epidemics on ‘Gros Michel’ (AAA), ‘Silk’ (AAB), ‘Pomme’ (AAB) and ‘Pisang Awak’
(ABB). Race 2 affects cooking bananas such as ‘Bluggoe’ (ABB) and some AAAA
tetraploids. Race 3 caused epidemics on Heliconia spp., while race 4, that is causing
high losses in Asia, but is still not present in America, is virulent to ‘Cavendish’ (AAA)
and susceptible varieties of race 1 and 2 (Ploetz and Pegg 2000, Pérez-Vicente and
Battle 2010a). The term “race” in Foc does not imply a defined, genetic relationship with
the host; they are simply strains that are pathogenic on the differential cultivars (Ploetz
1994).
The term “Vegetative Compatibility Groups” referred to here as VCG, is a fast
and easy way to distinguish Foc pathotypes (Puhalla 1985). Conceptually they are
strains which are identical at a particular set of loci but are capable of forming a stable
heterokarion (Leslie 1993) indicated by a dense growth where two colonies touch
(Puhalla 1985). Isolates in a VCG are often in the same race and usually share similar
geographic ranges and physiological characters. VCG’s provide a wealth of information
on the dissemination and evolution of this pathogen (Ploetz 1994, Ploetz and Pegg
2000). VCG’s for Foc were first reported by Puhalla in 1985 and 21 VCG’s are presently
described (Pegg at al. 1996). The VCG’s and races of Foc are shown in Table 1.
4
Table 1: Grouping of Fusarium oxysporum f. sp. cubense according VCG, race and geographical distribution.
Source: adapted by Pérez-Vicente and Batlle (2010b) & Ploetz and Pegg (2000).
VCG*
VCG Complex**
Race***
0120
0120 - 01215
1, 4
0121
0122
none
none
4
4?
Distribution by Countries****
South Africa, Australia, Brazil, Costa Rica,
Honduras, Jamaica, Indonesia, Guadaloupe, Canary
Islands, Malaysia, Taiwan.
Indonesia, Malaysia, Taiwan.
Philipines.
0123
0123a - 0123b
1
Philipines, Indonesia, Malaysia, Thailand, Vietnam.
0124
0124 - 0125 - 0128 - 01220
1, 2
0125
0124 - 0125 - 0128 - 01220
1, 2
0126
none
0128
0124 - 0125 - 0128 - 01220
1, 2
0129
01210
01211
01212
01213
01214
01215
01216
01217
01218
01219
01220
01221
none
none
none
none
01213 - 01216
none
0120 - 01215
01213 - 01216
none
none
none
0124 - 0125 - 0128 - 01220
none
4
1
4
?
T4
2
1, 4
T4
?
?
?
4?
?
1
Australia, Burundi, Brazil, Cuba, USA, Honduras,
India, Jamaica, Kenya, Malaysia, Malawi,
Nicaragua, Philipines, Thailand, Uganda, Tanzania,
Vietnam, Indonesia.
Australia, Brazil, Honduras, India, Jamaica, Kenya,
Malaysia, Malawi, Nicaragua, Philipines, Thailand,
Uganda, Tanzania, Vietnam, Zaire.
Honduras, Indonesia, Philipines.
Australia, Comores Islands, Cuba, Kenya, India,
Thailand.
Australia.
Cuba, USA.
Australia.
Kenya, Tanzania, Uganda.
Australia, Indonesia, Malaysia.
Malawi.
Costa Rica, Indonesia, Malaysia.
Australia, Indonesia, Malaysia.
Malaysia.
Indonesia, Malaysia, Philipines, Thailand.
Indonesia
Australia, India, Kenya, Thailand.
Thailand.
*VCG's: vegetative compatibility groups as defined in the text. **VCG complex: broad group of VCGs in which cross-compatibility or bridging
occurs. ***Race: physiological races as defined in the text. ****Distribution by country: data from various authors compiled by Pérez-Vicente
(2004).
1.2.3. Fusarium oxysporum f. sp. cubense Biology
The causal organism, Fusarium oxysporum f. sp. cubense, a Deuteromycete,
member of the Elegans section of Fusarium (Fungi Imperfecti), produce three types of
asexual spores: microconidia, macroconidia and chlamydospores (Leslie and
Summerell 2006). At first, 0- and 1-septate microconidia are produced in abundance
and are ovate or somewhat elongated (Wardlaw 1961). Later macroconidia are formed,
more than 95% of them are 3-septate, but a few are 4- or 5-septate (Wardlaw 1961).
Microconidia and macroconidia are formed from monophialidic or polyphialidic
conidiogenous cells (Burgess et al. 1994). The chlamydospores are usually formed
single or in pairs, but also may be found in clusters or in short chains (Leslie and
5
Summerell 2006). Composed of a hard and tick cell wall, their function is primarily
survival in soil (Burgess et al. 1994, Leslie and Summerell 2006). Once the soil is
infested with the pathogen, it cannot be planted with susceptible clones for up to 30
years (Ploetz 1994, Moore et al. 1995). Asexual reproduction in Foc is accomplished by
macroconidia and microconidia, while a sexual state of the fungus has never been
observed (Booth 1975, Cordeiro and Kimati 1997).
1.2.4. Infection and Dissemination
The pathogen is a soil inhabitant and establishment in host occurs by way of the
root system, with the most common site of penetration the young root tips (Wardlaw
1961, Cordeiro and Kimati 1997). The fungus invades the xylem vessels, and if not
blocked by vascular occluding responses of the host, advances into the corm, producing
microconidia within 2-3 days (Moore et al. 1995, Ploetz and Pegg 2000). The pathogen
ultimately causes terminal wilt. It then grows out of the xylem into the surround tissues,
forming many chlamydospores which are returned to the soil when the plant tissue
decays, thereby allowing the pathogen to endure long periods of dissecation and the
absence of a host (Moore et al. 1995, Ploetz and Pegg 2000). The most common
means by which Foc is disseminated is in the transport of infected corms (Su et al.
1986, Moore et al. 1995, Ploetz and Pegg 2000, Cordeiro and Matos 2003, Davis 2005,
OIRSA 2008, Ploetz 2009, Pérez-Vicente and Pocasangre 2010). However,
dissemination can also occur through spread in soil adhering to farm implements and
machinery (Moore et al. 1995, Ploetz and Pegg 2000, Cordeiro and Matos 2003, OIRSA
2008, Pérez-Vicente and Pocasangre 2010). Water is also a relevant dissemination
path. Attention must be taken regarding spread in the water from surface run-off,
irrigation and floods (Su et al. 1986, Moore et al. 1995, Cordeiro and Matos 2003,
Ploetz and Pegg 2000).
1.2.5. Fusarium Wilt Symptoms
Symptoms in Foc diseased plants can be observed internally and externally. The
internal symptoms are characterized by reddish to dark brown discoloration of the host
vascular system, first into the roots and corms, and then advancing to the pseudostem
6
(Wardlaw 1961, Moore et al. 1995, Ploetz and Pegg 2000). Through a transversal plant
cut, more intense discoloration may be observed on the outermost sheaths of the
pseudostem in direction to the innermost or young leaf sheaths (Wardlaw 1961). The
first external symptoms of Foc disease in banana are a yellowing of the oldest leaf
reaching progressively the youngest leaves by a vivid band along the margin and
subsequently spreading inwards towards the mid ribs. Leaves gradually collapse at the
petiole and hang down to form a “skirt” of dead leaves around the pseudostem
(Wardlaw 1961, Su et al. 1986, Moore et al. 1995, Cordeiro and Kimati 1997, Ploetz and
Pegg 2000, Cordeiro and Matos 2003). Longitudinal splits at the base of pseudostem
are often used to detect Foc diseased banana plants (Wardlaw 1961, Cordeiro and
Kimati 1997, Cordeiro and Matos 2003, David 2005). Internal and external symptoms
can be seen in Figure 2.
Figure 2: (A) Advanced Fusarium Wilt external symptom in Gros Michel (AAA). (B) Internal symptom of pseudostem
vascular tissue discoloration in Gros Michel (AAA). Photos: Paulo S. F. Lichtemberg.
7
1.2.6. Fusarium Wilt Occurrence in Central America
The banana cultivar ‘Gros Michel’ (AAA) is still cultivated by smallholders in
Central America due the preference of consumers for the fruit flavor throughout those
countries (Silagyi and Pocasangre 2003). ‘Gros Michel’ (AAA) also produces excellent
fruit that are more durable than those of ‘Cavendish’ cultivars (Ploetz et al. 2007).
Currently many smallholders produce intercropped ‘Gros Michel’ (AAA) with coffee,
cocoa and cassava (Silagyi and Pocasangre 2003, Pocasangre 2008, Pocasangre
2009). The presumed absence of ‘Gros Michel’ (AAA) monoculture probably delayed
detection of Fusarium Wilt in the above situation (Ploetz 1994). However Foc disease
remains a serious production constrain where susceptible cultivars are grown (Ploetz
1994, Moore et al. 1995, Silagyi and Pocasangre 2003, Pocasangre 2008, Pocasangre
2009, Lara et al. 2009). The phylogenetic race 4, still not present in the American
continent, represents a serious risk to banana production, considering the capability to
infect and kill 80% of all banana genotypes produced in Latin America and the
Caribbean (OIRSA 2008, Dita et al. 2009, Pocasangre and Pérez-Vicente 2009, Ploetz
2009). The entrance of race 4 would strongly impact socially and economically the
banana food chain in Latin America and Caribbean (Pocasangre and Pérez-Vicente
2009). In absence of control methods against race 4, smallholders in affected areas
would be often forced out of banana and resort to the production of other crops which
are nutritionally poorer, often less economically advantageous and less environmentally
friendly than bananas (Swennen et al. 2000).
1.2.7. Control Methods
In general, effective chemical control measures do not exist (Ploetz and Pegg
2000, Cordeiro and Matos 2003, Pocasangre 2009). All chemical control implemented
in the past has been shown to have adverse effects due the broad spectrum of activity
and negative effects on soil microorganisms (Moore et al 1995, Goés and Moretto 2001,
Cordeiro and Matos 2003). However, Nel et al. (2007) demonstrated an 80.6%
reduction in disease with the usage of Benomyl and the demethylation applied as root
dip treatment. In Central America the shifting production of the crop and production as
an annual crop became strategies to avoid the problems cause by Foc. However these
8
strategies only allow short term production before new infestations occur (Pocasangre
2009).
The use of genotypes tolerant and/or resistant to Foc disease is the most
effective management tool (Su et al. 1986, Moore et al. 1995, Cordeiro and Kimati
1997, Goés and Moretto 2001, Cordeiro and Matos 2003). The problem at the moment
is the absence of cultivars which offer the organoleptic quality presented in the
‘Cavendish’ and ‘Gros Michel’ subgroup in case of race 4 infestations. Micropropagated
plant material are the most reliable source of clean material, since they are also free of
bacteria, nematodes, and other fungal pathogens. Therefore, plantlets should be used
whenever is possible (Wardlaw 1961, Su et al. 1986, Moore et al. 1995, Ploetz and
Pegg 2000). However if Fusarium wilt is in the soil, these approaches are only short
lived.
The usage of endophytic antagonistic microorganisms represents an alternative
as biocontrol agent that can be used to decrease the level of disease incidence,
improve nutrition and plant resistance (Pocasangre et al. 2000). Promising results have
been obtained with the biological control of Fusarium Wilt, through the use of nonpathogenic strains of Fusarium oxysporum and Trichoderma spp. (Pocasangre 2008).
Also recent work with endophytic bacteria of the genus Pseudomonas spp. and Bacillus
spp. from the banana rhizosphere has demonstrated biocontrol activity to the wilt
pathogen under controlled conditions (Pocasangre 2008). The future use of biologicalchemical combinations of endophytes and commercial pesticides applied on the seed or
seedling also could lead to synergistic effects on one or multiple disease causing
agents. Instantaneous suppression on pathogenic organism by the pesticide could be
supported by the biological control agent during the crop cycle (Backman and Sikora
2008). Disease suppressive soils were found in several different locations as Santa
Marta in Colombia (Wardlaw 1961), Canary Islands, Australia and South Africa (Moore
et al. 1995). These types of soils allow production to continue in spite of the presence of
the pathogen. Suppressive soils may be more widely spread than is currently
appreciated (Ploetz 1994, Ploetz and Pegg 2000).
While chemical, tolerant and resistant varieties and biological control are still
improving management against specific plant diseases, the most important method of
9
Foc control is quarantine. Exclusion and eradication must be stress in the forefront in
attempts to avoid disease import and later outbreaks (Wardlaw 1961, Moore et al. 1995,
Cordeiro and Kimati 1997, Davis 2005, Dita et al. 2009, Molina 2009).
In the present investigations it was hypothesis that (1) Fusarium Wilt is affecting
bananas produced in association with coffee under tree shade in Costa Rica and
Nicaragua study areas (2) Incidence of Foc differs among countries and zones (3)
Growers have knowledge and methods to manage the disease and (4) there are
differences in virulence among isolates recovered in a region.
1.3.
Objectives
(1)
To determing the occurrence, incidence and severity of Fusarium Wilt
disease (Fusarium oxysporum f. sp. cubense Foc) affecting banana when grown
with coffee under forest tree shading at study areas in Costa Rica and
Nicaragua.
(2)
To evaluate the traditional knowledge concerning Fusarium Wilt disease.
(3)
To conduct greenhouse pathogenicity tests using the Costa Rican Foc
isolates.
(4)
To determine isolates virulence and their effect on plant growth in the
greenhouse.
10
Chapter 2 Fusarium Wilt Incidence and Severity
CHAPTER 2
2.
2.1.
Fusarium Wilt Incidence and Severity
Introduction
Research on Fusarium Wilt went through two important eras. The “Gros Michel
era” started with the first comprehensive review of the disease by Stover in 1962 who
discuss the possible origins and research associated to ‘Gros Michel’ epidemics in the
American tropics. Although the disease was widely reported around the world by the
early 1900s, little sustained research was conducted until the middle of the century
(Moore et al. 1999a). During the “Cavendish era” the research was focused on the
diversity of the pathogen and not into the disease (Moore et al. 1999a). Consequently
Foc disease continued to be a problem in dessert bananas produced by smallholders
(Buddenhagen 1990; Moore et al. 1995).
Understanding the present status of Foc disease in Costa Rican and Nicaraguan
smallholders banana plantations is essential to estimate potential losses. This
knowledge permits the detection of diseased-free areas and avoiding entrance of new
strains where a limited diversity already exist (Moore et al. 1999a). In this chapter the
occurrence, incidence and severity of Fusarium Wilt was examined for study areas in
CR and NIC, In addition smallholder profiling was conducted to gain a better
understanding of the possible effect of production method used by the farmers on
disease.
2.2.
Experimental Site
The study area included two zones in Costa Rica (09o56’ North, 84o5’ West) and
in Nicaragua (13o0’ North, 85 o0’ West). Turrialba city in Cartago province received the
zone-1 with 14 farms, in Alto Quetzal, Bajo Pacuare and Paso Marco communities and
in zone-2 with 16 farms, in San Juan Sur, San Juan Norte and Chitária communities.
In Nicaragua, Jinotega city received the zone-1 with 15 farms in Monterrey
community and San Ramón city received the zone-2 with 15 farms in Yassica Sur
community (Figure 3).
11
Figure 3: Experimental sites of Costa Rica and Nicaragua in Central America. Map left: Centro America continent.
Map up right: Nicaragua, orange dots: study zones and Managua, black star: UNAN laboratory. Map down right:
Costa Rica, orange dots: study zones and San Jose, black star: CATIE laboratorio. Source: CCAD, elaborated by
Natalia Estrada Carmona.
2.3.
Material and Methods
A total of 30 farms were accessed baseline and research studies conducted in
CR and NIC. Support was given by the local partners: University of Costa Rica (UCR),
Turrialba Organic Producers Association (APOT) in Costa Rica and the Nicaraguan
Autonomy National University (UNAN) in Nicaragua.
2.3.1. Farm Selection
Farm selection began with a 150 to 200 growers list, prepared by local partners
with information on: names, addresses, farm size, and area planted to banana, coffee
and trees. The first screening was performed by selecting farms of up 15 ha and having
banana and coffee growing under trees on at least 1 ha. The second screening was
conducted by randomly choosing 30 farms.
2.3.2. Plot Setting
Prior to selecting the test plots the shape of the area where banana was grown in
association with coffee shaded by trees was determined (Figure 4A-B). After examining
12
S
the lot profile, the number of potential plots was estimated for further random selection
(Figure 4C). Plots
lots boundaries were marked using yellow plastic tape with a cross at the
middle creating 4 subsectors for orientation (Figure 4D).
Figure 4: Plot setting stages. (A) Total Farm;
Farm (B) Agroforestry lot and size; (C) Agroforestry
groforestry lot with amount of
possible plots; (D) Plot
lot scheme showing trees (red), coffee (green) and banana (yellow).
2.3.3. Base Line Study
2
During the base line study (BLS) 30 farms with 2 plots of 25x25 meters (625m
(
)
each were selected for farm characterization. The collected data were: banana, coffee
and other tree density; number of banana by cultivar; number of banana pseudostem
per mat; geographic coordinates; inclination, altitude
altitude,, trees names and number of
Fusarium Wilt diseased bananas.
bananas
‘Gros Michel’ sub-group
group bananas exhibiting external and internal Fusarium Wilt
symptoms were counted for Foc incidence (Subramaniam et al. 2006). The internal
symptom was confirmed present by observation of brownish strips in the vascular tissue
by exposing internal stem tissue by superficial cutting.
% 2.3.4. Detailed Study
The Detailed Study (DS) included Foc disease severity grading and results for
vascular tissue sampling as seen in the “Field Form – FF001” (Annex 1).
1)
13
The Foc severity was graded according to the International Network for the
Improvement of Banana and Plantain (INIBAP) technical guideline (Orjeda 1998), using
a 1 to 5 scale for external symptoms such as yellowing and wilting (Table 2) and 1 to 6
for internal symptoms of discoloration (Figure 5).
Table 2: Scale for rating external symptoms caused by Fusarium oxysporum f. sp. cubense.
Grade
External Symptoms (Yellowing and Wilt)
1
Absence of Yellowing and Wilt.
2
Yellowing and Wilt in the lowest leaves (old leaves).
3
Yellowing and Wilt extensive.
4
Yellowing and Wilt in the upper leaves (young leaves).
5
Yellowing and Wilt severe (all leaves).
Source: Orjeda (1998)
Figure 5: Scale for rating the internal symptom caused by Fusarium oxysporum f. sp. cubense (Orjeda 1998).
The vascular tissue sampling was carried on a maximum of 3 sub-samples per
plot, whereby pseudostem and corm samples were taken separately. The pseudostem
sample was removed by one longitudinal “L” shaped cut at 50cm height (Figure 6A)
whereas the corm sample was taken from the top of the rhizome (Figure 6B). Small
samples were collected and packed in paper bags to avoid decay and then processed
in the lab as soon as possible.
14
Figure 6: Vascular tissue sampling. (A) pseudstem vascular tissue sampling location; (B) Corm vascular
tissue location. Red arrows indicate sampling locations. Source: adapted from Soto (2008).
2.4.
Statistics
Pearson correlation coefficient test (p<0.05) was used to identify relationship
between the Foc disease incidence and farm variables such as: banana-coffee-tree
density, ‘Gros Michel’ density, banana stem average, number of leguminous trees,
inclination and altitude. The statistical analysis was conducted with the software Infostat
® version 2009e (Di Rienzo 2009).
2.5.
Results
2.5.1. Farms Profile
Smallholders total farms size and associated banana and coffee area in CR
correspond to 8.5 and 2 ha-1 respectively (Figure 7A). In NIC total farm size and
associated banana and coffee area were 5.5 and 4.2 ha-1 respectively (Figure 7B).
Banana and coffee area in CR and NIC correspond respectively to 23% and 73% of
total farm size (Figure 8). The farms producing banana in CR and NIC were found at an
15
altitude of 900 and 865 meters above sea level respectively (Figure 9A), and field
inclination was 21% in CR and 15% in NIC (Figure 9B).
Total Plant density in CR and NIC was 5,104 and 5,357 plants per hectare
respectively. In CR the ratio of coffee:banana:shade trees was 83% coffee (4,218
plants/ha), 11% banana (573 plants/ha) and 6% trees (313 plants/ha) while in NIC 90%
was coffee (4,818 plants/ha), 7% banana (355 plants/ha) and 3% trees (184 plants/ha)
(Figure 10). ‘Gros Michel’ covered 44% (252plants/ha) and 86% (305 plants/ha) of the
total bananas grown in CR and NIC respectively (Figure 11). Annual banana yield per
hectare in CR and NIC according to the growers was 200 and 233 bunches/ha-1
whereas coffee production was estimated at 10 and 25 gold coffee tons/ ha-1
respectively. Annual banana production as estimated by the growers is shown in Figure
12A and coffee production in Figure 12B. Gold coffee means the seed dried without
peel and ready for processing.
A
B
20
20
n=30
µ=8.5
µ=
µ=2
µ=
15
10
5
0
Nicaragua Farm Size (ha-1)
Costa Rica Farm Size (ha-1)
n=30
µ=5.5
µ=
µ=4.2
µ=
15
10
5
0
CR total size
CR associated size
NIC total size NIC associated size
Figure 7: (A) Total farm size and associated banana and coffee lot size in Costa Rica. CR=Costa Rica. (B) Total farm
size and associated banana and coffee lot size in Nicaragua. NIC=Nicaragua.
16
23%
73%
77%
27%
Costa Rica
Nicaragua
Banana associated with coffee - 23%
Other land usage - 77%
Banana associated with coffee - 73%
Other land usage - 27%
Figure 8: Smallholder land usage in Costa Rica and Nicaragua. Other forms of land usage in Costa Rica= pasture,
sugar cane, root and grain production. Other forms of land usage in Nicaragua= grain production. N=30.
A
50
n=30
B
n=30
1400
1200
Inclination (%)
Altitude (m.a.s.l.)
40
1000
800
30
20
10
0
600
µ=900
Costa Rica
µ=865
Nicaragua
µ=21
Costa Rica
µ=15
Nicaragua
Figure 9: (A) Altitude of Costa Rica and Nicaragua smallholders. (B) Inclination of Costa Rica and Nicaragua
smallholders.
17
4,218 plt/ha
83%
313 plt/ha 6%
573 plt/ha
11%
4,818 plt/ha
90%
Costa Rica
184 plt/ha 3%
355 plt/ha
7%
Nicaragua
Tree
Coffee
Banana
Tree
Coffee
Banana
Figure 10: Tree, coffee and banana participation per hectare (density and share) in Costa Rica and Nicaragua
smallholders plantations. Plt=Plants.
Banana Type Participation (%)
100
80
252 plt/ha
43%
305 plt/ha
86%
60
40
20
321 plt/ha
57%
50 plt/ha
14%
0
Costa Rica
Nicaragua
Other banana cultivars
Gros Michel (AAA) cultivar
Figure 11: Banana type participation per hectare (density and share) in Costa Rica and Nicaragua smallholders.
Plt=Plants, Other banana cultivars=mainly ‘cavendish’ and ‘cooking banana’ subgroups.
18
A
n=30
n=27
800
600
400
200
0
µ=200
µ=233
Costa Rica
Nicaragua
100
Coffee production (gold seed/yr/ha-1)
banana production (bunch/yr/ha-1)
1000
B
n=30
n=29
µ=10
µ=25
80
60
40
20
0
Costa Rica
Nicaragua
Figure 12: (A) Banana production in Costa Rica and Nicaragua smallholders (grower estimative). (B) Coffee
production in Costa Rica and Nicaragua smallholders (grower estimative). Gold seed = coffee fruit without pulp.
2.5.2. Costa Rica
In CR, Foc disease was affected 10 of 30 surveyed farms, or 33% of the fields
sampled. Foc disease in zone-1 was detected in 6 of 14 farms (43%) and in zone-2
affected 4 of 16 farms (25%) (Figure 13). Disease distribution per zone is show in the
illustrative map (Figure 14).
Foc disease incidence was 7.3% for CR, 12.7% for CR zone-1 and 2.6% for CR
zone-2 (Figure 15). There was no significant (p<0.05) correlation between Foc
incidence and banana, coffee, shade trees density, field altitude and inclination, number
of ‘Gros Michel’ and banana pseudostem average as demonstrate in the Table 3. There
were also no statistical differences (p=0.176) for incidence among zones-1 and -2
(Table 5B).
Fusarium Wilt severity in CR attributed at the time of sample collection, reveal
external symptoms of 3.4 for yellowing and 3.3 for wilt and internal symptom of 2.8 for
corm discoloration (Figure 16A). In zone-1 yellowing was estimated as 2.8 for yellowing,
2.9, for wilt and 1.8 for corm discoloration (Figure 16B), while in zone-2 the results for
yellowing were 3.8 for wilt 3.7 wilt and 3.75 for corm discoloration (Figure 16C).
19
Country
Foc positive
10 farms (33%)
Foc Negative
20 farms (67%)
Zone 1
Zone 2
Foc Positive
6 farms (43%)
Foc Negative
8 farms (57%)
Foc Positive
4 farms (25%)
Foc Negative
12 farms (75%)
Figure 13: Occurrence of Fusarium Wilt in Costa Rica and respective zones (number and percentage).
Foc=Fusarium Wilt (Fusarium oxysporum f. sp. cubense). Country=Costa Rica study zones.
Figure 14: Map of farms localization and Fusarium Wilt occurrence in zones 1 and 2 of Costa Rica. Map left: Costa
Rica and study zones. Map up right: Study zone 1, farms on Revetazón River domain. Map lower right: Study zone 2,
Farms on Pacuare River domain. Color dots = farm plots. Numbers = lots. Black ring = Fusarium Wilt diseased lots.
Source: Comissión Centroamericana de Ambiente y Desarrollo (CCAD), Elaborated by: Natalia Estrada Carmona.
20
Fusarium Wilt Incidence (%)
20
15
10
5
7.31
12.65
2.63
Costa Rica
CR zone 1
CR zone 2
0
Figure 15: Fusarium Wilt plant incidence in Costa Rica and
corresponding zones. CR=Costa Rica. Bars indicate Standard Error of
the Mean.
Table 3: Statistical correlation of Fusarium Wilt
incidence and field data measures in Costa Rica.
Incidence
(p)
(r)
Banana Density
0.148
0.27
Coffee Density
0.758
-0.059
Tree Density
0.289
-0.2
Gross Michel Density
0.23
-0.226
o
0.302
-0.195
n leguminous trees
o
0.295
-0.198
Altitude
0.41
-0.11
Inclination
0.12
0.21
n banana stems
Pearson correlation (Significance 0.05)
21
A
B
C
6
6
7
5
5
6
4
4
3
3
2
2
5
4
3
2
1
1
0
µ=3.4
Costa Rica
µ=2.75
µ=3.83
zone 1
zone 2
0
1
µ=3.25
Costa Rica
Yellowing
µ=2.88
zone 1
µ=3.67
zone 2
Wilt
0
µ=2.8
µ=1.75
Costa Rica
zone 1
µ=3.75
zone 2
Discoloration
Figure 16: External and Internal Disease Severity caused by Fusarium Wilt in banana plants in Costa Rica
smallholders plantations. (A) Yellowing. (B) Wilt. (C) Internal corm discoloration.
2.5.3. Nicaragua
Foc disease symptoms were detected in 7 of 30 surveyed farms, representing
23% of the total sample. In NIC zone-1, 7 of 15 farms (47%) were diseased whereas in
zone-2 the plants were Foc disease-free (Figure 17). The disease distribution map is
presented in Figure 18.
In NIC Foc disease incidence was 1.12%. The NIC zone-1 had showed disease
incidence of 2.24% while the zone-2 was disease-free (Figure 19). Foc incidence was
not significantly (p<0.05) correlated to the field measurements such as: banana, coffee,
shade trees density, field altitude and inclination, number of ‘Gros Michel’ and banana
pseudostem average (Table 4). Zone-1 and -2 differ statistically (p=0.0073) concerning
Fusarium Wilt incidence (Table 5C).
Fusarium Wilt severity at the time of sample collection, was index at 2.6 external
yellowing and 2.5 for wilt. Pseudostem discoloration was indexed at 1.2 (Figure 20).
22
Nicaragua (country)
Foc Positive
23% (7 farms)
Foc Negative
77% (23 farms)
Nicaragua (zone 1)
Nicaragua (zone 2)
Foc Positive
47% (7 farms)
Foc Negative
53% (8 farms)
Foc Negative
100% (15 farms)
Figure 17: Occurrence of Fusarium Wilt in Nicaragua and respective zones (number and percentage). Foc=Fusarium
Wilt (Fusarium oxysporum f. sp. cubense).
Figure 18: Map of Farms Localization map and Fusarium Wilt occurrence in zone-1, Jinotega and zone-2, San
Ramón in Nicaragua. Map left: Nicaragua, Farms of Jinotega and San Ramón districts. Map up right: District of
Jinotega, community of Monterrey. Map lower right: District of San Ramón, community of Yassica Sur. Color dots =
farm plots. Black rings = Fusarium Wilt diseased lots. Source: Comissión Centroamericana de Ambiente y Desarrollo
(CCAD), Elaborated by: Natalia Estrada Carmona.
23
Fusarium Wilt Incidence (%)
4
3
2
1
1.12%
2.24%
Nicaragua
NIC zone 1
0
Figure 19: Fusarium Wilt plant incidence in Nicaragua and zone-1.
NIC=Nicaragua. Bars indicate Standard Error of the Mean.
Table 4: Statistical correlation of Fusarium Wilt
incidence and field data measures in Nicaragua.
Incidence
(p)
(r)
Banana Density
0.414
0.155
Coffee Density
0.58
-0.105
Tree Density
0.307
0.193
Gross Michel Density
0.202
0.24
n banana stems
0.678
0.079
no leguminous trees
0.289
0.2
Altitude
0.07
-0.34
Inclination
0.19
0.25
o
Pearson correlation (Significance 0.05)
24
5
4
3
2
1
µ=2.6
µ=2.5
Yellowing
Wilt
µ=1.2
0
Discoloration
Figure 20: Severity of external and internal symptoms caused by Fusarium Wilt in banana plants of Nicaragua
smallholders.
Fusarium Wilt incidence between Costa Rica and Nicaragua did not differ
statistically (p=0.098) as shown in Table 5A.
Table 5: Statistical analysis of: (A) Fusarium Wilt incidence among countries. (B) Fusarium Wilt incidence within
Costa Rica zones. (C) Fusarium Wilt incidence within Nicaragua zones.
A
B
Coutries
n
Costa Rica
30
Nicaragua
30
Fusarium Wilt
Incidence
C
Costa Rica Zones
n
7.31 a
Zone 1
14
1.12 a
Zone 2
16
Fusarium Wilt
Incidence
Fusarium Wilt
Incidence
Nicaragua Zones
n
12.65 a
Zone 1
15
2.24 a
2.63 a
Zone 2
15
0b
Different letters means significative differences (p<=0.05)
2.6.
Discussion
The presence of Foc in banana plants has been extensively reported for Central
America (Wardlaw 1961, Stover and Simmonds 1987, Moore et al. 1995, Ploetz 2005a).
Nevertheless only a few studies examined Foc incidence in detail for these countries
(Pocasangre 2009). Additionally there is a lack in bio-ecological studies concerning Foc
disease in the region (Rodriguez et al. 2002).
25
Foc occurrence and incidence in CR farms decreased within a period of 6 years.
A survey performed in 2003 revealed that 90% of 11 sampled farms in Turrialba city
were Foc infested (Silagyi and Pocasangre 2003) with diseased plant incidence of 16%
(Pocasangre and Lichtemberg 2010). In 2009, Foc disease occurrence on farms
sampled dropped to 33% and Foc plant disease incidence to 7.3%, representing
respectively reductions of 57% and 8.7%. The decrease in Foc disease was mainly
explained by the replacement of susceptible by resistant varieties from ‘Cavendish’ subgroup. According to the growers the replacement of ‘Gros Michel’ cultivars was more
frequent in recent years due lower levels of resistance to diseases. Replacement was
not favored by the growers due to the fact that much better prices are obtained on the
local market for ‘Gros Michel’. This demonstrates a dramatic change whereby growers
are becoming more dependent on a different and not wanted dessert banana subgroup.
Severity field data collected during the present study must be carefully interpreted.
Considering the differences in plant developmental stages and the period of pathogen
incubation these results can only serve as base line data to support more detailed
futures surveys.
In NIC Foc disease occurred exclusively in zone-1 Monterrey community in
Jinotega, whereas zone-2, Yassica Sur community in San Ramón was free of Foc
disease. Cultural aspects might explain the differences. In northern NIC banana is not
the main crop and the exchange of planting material is not a common practice between
farmers. In addition, Yassica Sur growers have a greater diversity of banana cultivars in
their fields and these are geographically isolated from Monterrey, two important facts
that limit Foc dissemination. Historical development of banana industry in NIC certainly
is related to the incidence observed. Banana production in NIC is relatively new in
Central America (Aguilar 1995, Soto 2008). In 1935, Foc destroyed a huge amount of
susceptible banana in plantations in Central America and during the same period
banana industries closed their doors for approximately 20 years because of the
Sandinist Revolution (Soto 2008). This interruption in expansion of the banana industry
certainly affected disease dissemination. In addition, the northern region of NIC is not
considered to be a traditional banana production region (Aguilar 1995). Coffee is the
main crop cultivated, and banana is used traditionally for self consumption and/or
26
shade. There are no reports of past surveys for the northern NIC region, however, it has
been reported that in the southwest Pacific region Foc disease incidence in ‘Bluggoe’
cultivars averaged 9.9% of sampled plants (Gongora and Narvaes 1994), 2.4% to 6%
during the wet season in December 2001, increasing to 8.4% in dry season, February
2002 (Dolmuz et al. 2002). In the same southwest region in Rivas Department, 36% of
sampled farms were Foc disease positive (Rodriguez et al. 2002). Even when
considering differences in epidemiology among Foc races the data shows similar values
of incidence during these years. The level of Foc severity in NIC demonstrated
extensive yellowing and wilting for external symptoms and corms completely clean with
no vascular tissue discoloration for internal symptoms. These results are again only
preliminary base line data since it is important to consider that severity also varies
according inoculum density in soil in a particular region (Ploetz and Pegg 2000).
Worldwide, studies performed in Malaysia showed that small farms with less than
2 hectares had high disease occurrence of up to 50% (Jamaluddin et al. 1999).
Therefore, CR and NIC where disease occurrence was 20 to 30% can be considered
moderately affected. In Bangladesh field studies reported disease incidence ranging
from 4 to 24% using the number of plants wilted per study region which they considered
high (Hossain and Rashid 1999). In the zones surveyed in CR and NIC average Foc
incidence of 2.6% (CR zone-2) to 12.65% (CR zone-1) and 0% (NIC zone-2) to 2.24%
(NIC zone-1) once more placing the Central American countries under the Asiatic
incidence rates.
The absence of a correlation between disease incidence and field measured data
demonstrated that other parameters such as soil, fertility or local climatic conditions may
be influencing Foc occurrence in banana plants (Simmonds 1959). Despite the fact that
the incidence of Foc in CR was slightly greater than in NIC, the data was not statistically
different and indicates that the field measured data above mentioned do not play a role
in disease and that similar disease management strategies could be used in both
regions to reduce or retard Foc disease spread and development.
27
2.7.
Conclusion
(a)
In Costa Rica and in Nicaragua study areas, Fusarium Wilt is affecting 33
and 23% of banana farms associated with coffee under tree shade respectively.
(b)
Fusarium Wilt incidence does not differ statistically among countries.
However within the Nicaragua studied areas Fusarium Wilt disease incidence
differed among Monterrey and Yassica Sur zones. In Costa Rica there was no
difference between the studied zones.
(c)
In Nicaragua Fusarium Wilt occurrence is restricted to one of two
surveyed zones.
(d)
In the present survey, Yassica Sur or zone-2 in Nicaragua was found
disease-free. It may be related to the smaller density of susceptible varieties and
greater diversity of resistant varieties.
2.8.
Recommendation
(a)
Carry out studies in the same surveyed farm areas and include additional
factors that might be correlated with Foc incidence, such as: physical and
chemical soil parameters, temperature and rainfall parameters and plant age and
root health condition.
(b)
Consider the Nicaraguan zone-2 for suppressiveness studies.
28
Chapter 3 The Fusarium Wilt Traditional Knowledge
CHAPTER 3
3.
3.1.
The Fusarium Wilt Traditional Knowledge.
Introduction
The last few decades have been characterized by the emergence of new
infectious diseases and re-emergence of old infectious ones (Wilcox and Ellis 2006).
Furthermore, most studies, old and recent, have tended to look at the impact of disease
on food security by focusing on a single disease (Rugalema et al. 2009). Before
chemical control become available, one of the phytosanitary cornerstones of agriculture
was cultural practices that included: crop rotation, increasing diversity of plants, shade
management and others (Schroth et al. 2000). Understanding grower’s traditional
agricultural knowledge must be an early step of in a research program. By traditional
knowledge we mean the cultural and technical knowledge that growers in a specific
area have and use. Growers inherit part of this knowledge from their ancestors and
build upon other sources of information and their own experiments and experience
(Morales and Perfecto 2000). The richness of this knowledge is enormous (Altieri 1984).
By this mechanism it is possible to understand what farmers are doing, why they are
doing it in a particular way, and what is required if a new technology is to be accepted
by growers. This information facilitates disease control strategies advocated by experts
and policy makers, for example, the Foc race 4 exclusion campaign of the American
continent being carried out by OIRSA.
The objective of the present studies was to document traditional farm practices
regarding Foc disease knowledge and management. A semi-structured questionnaire
was applied during interviews which covered topics related to: banana crop importance
and management, disease occurrence, dissemination and control. In this chapter and
attempt was made to demonstrate the existence of a relationship between traditional
knowledge and Fusarium Wilt incidence in smallholder field with banana growing
associated with coffee under tree shade. The information acquired here might be useful
to support decisions made in future programs dealing with disease control in such
growing areas in Costa Rica, Nicaragua and elsewhere.
29
3.2.
Experimental Site
The experimental site used for the traditional knowledge evaluation was the
same as the farm characterization and the incidence study. Growers of Alto Quetzal,
Bajo Pacuare, Paso Marco, Chitaria, San Juan Norte and San Juan Sur communities in
CR and Monterrey and Yassica Sur communities in NIC where interviewed.
3.3.
Materials and Methods
Throughout the BLS and DS, 60 interviews were performed with growers of the
surveyed farms, with 30 interviews performed in CR and another 30 in NIC. One
preliminary “small talk” was conduct before the interview that was aimed at introducing
the grower to the research, myself, to determine the general knowledge of the individual
grower, and to generate trust between the interviewer and interviewees (Briggs 1997).
The questionnaire had a semi-structured format, which made it possible to receive
complete answers and at the same time allow the interviewed grower his free opinion
on topics which they might have more familiarity (Dillon 1997).
The interview format contain an introductory message to clarify to the growers
what the aim of the visit followed by 5 question sections: (1) farm section, 4 questions,
(2) coffee section, 4 questions, (3) banana section, 6 questions, (4) banana disease
section, 8 questions, and (5) banana seed management, 4 questions (Annex 2). The
interviews were performed at the grower house or at the field according to the growers
preference and availability. The time required to complete the interview was
unpredictable, the interview was considered finished when the topics were totally
covered or disinterest from the grower side was evident.
3.4.
Statistics
Questions were analyzed by the Frequency of Answers and statistical differences
verified by Pearson’s Chi-Square Test. Pearson Correlation Coefficient Test was used
to find relationships among answers on Foc disease topics and the current disease
incidence status, and was demonstrate by Contingence Table and Correspondence
Analyze Graphics. The softwares Infostat ® version 2009e and SPSS 16.0 for windows
were used.
30
3.5.
Results
3.5.1. Banana Importance
In CR, banana was considered by 60% of the growers to be the most important
crop. Coffee represented the most important crop for 30% of growers and 10% of
pointed to other activities as most important.In NIC, banana production was considered
to be most important by only 10% of the growers whereas 76.7% named coffee as most
important. Other activities represented 6.7% and another 6.7% gave no answer (Figure
21A). Significant differences (p<0.0001) among answers were found, according Persons
Chi-square.
When asked about the reasons why banana is important, 46.7% of CR growers
declared monthly income as the main reason. Stable prices and additional income and
no specific reason were given in 10%, 13.3% and 30% of the cases respectively. In NIC
additional income is mentioned by 36.7% of growers, monthly income, stable price and
no reason correspond respectively to 3.3%, 6.7% and 53.3% (Figure 21B). Significant
differences (p<0.0001) among answers were found, according Persons Chi-square.
A
B
Most Important Crop (%)
10
6.7
6.7
80
30
76.7
60
40
20
0
60
10
CR
NIC
Banana
Coffee
Other activity
No answer
100
Banana Crop Importance (%)
100
80
30
53.3
60
13.3
10
6.7
40
20
46.7
36.7
3.3
0
CR
NIC
Monthly income
Aditional income
Stable price
No reason
Figure 21: Interview results about ‘crop importance’. (A) “Which crop do you consider the most important?” other
activity means: animal production, root crops and non-agricultural activity. (B) “Why Banana crop is important?”
CR=Costa Rica, NIC=Nicaragua.
31
3.5.2. Banana Management
Fertilization management in banana was performed by 36.7% of growers in CR
and 3.3% of NIC growers (Figure 22A). Significant differences (p=0.001) in the usage of
fertilizers was found, according Persons Chi-square. Of the growers performing
fertilization management in CR; 27.3% applied K-Mag (K2SO4.2MgSO4), 27.3% NPK,
18.2% chicken manure, 9.1% compost of coffee remnants, 9.1% calcarium and 9.1%
applied a combination of K-mag, compost and microelements. In NIC they only applied
product is Urea (Figure 22B). No significant differences in the types of fertilizers used
were found.
A
B
100
80
63.3
96.7
60
40
20
36.7
3.3
0
CR
Fertilization Management Type (%)
Fertilization Management (%)
100
9.1
9.1
80
60
9.1
18.2
100
40
27.3
20
27.3
0
NIC
Yes
No
CR
NIC
K-mag
NPK
Chicken manure
Calcarium
Compost
Compost K-mag microelements
Urea
Figure 22: Interview results about ‘fertilization management’. (A) “Do you perform fertilization management on
banana?” (B) “What fertilizer do you apply on banana?” K-mag=Potassium and Magnesium Sulfate and Sulfur.
NPK=Nitrogen, Potassium, Phosphorus. CR=Costa Rica. NIC=Nicaragua.
Concerning banana crop management, traditional pruning was performed both in
CR and NIC. The share of growers which do not perform any management in CR and
NIC correspond respectively to 10% and 30% (Figure 23A). There were no significant
differences regarding type of management. Growers which had received training on
32
banana production and disease management in CR and NIC representes 70% and
26.7% respectively (Figure 23B). There was a significant difference (p=0.001) in the
number of growers which were trained in banana and disease management.
A
B
100
100
13.3
80
30
23.3
60
33.3
13.3
40
10
20
30
0
CR
30
73.3
70
26.7
80
6.7
6.7
10
3.3
10
Banana Training (%)
Banana crop management (%)
10
60
40
20
0
NIC
Flower-leaf-sucker pruning
Flower-leaf-sucker pruning + plant orientation
Flower-leaf-sucker pruning + plant support
Leaf pruning
Sucker pruning
Leaf-sucker pruning
no management
CR
NIC
Trained
Not trained
Figure 23: Interview results about ‘banana management and training’. (A) “What crop management is performed on
banana?” (B) “Have you received training on banana production and disease management?” CR=Costa Rica.
NIC=Nicaragua.
3.5.3. Fusarium Wilt (Foc) Traditional Knowledge
When growers were asked if they knew of the Foc disease, 43.3% in CR and
10% in NIC answered the question positively (Figure 24A). After that, growers were
asked to describe the symptoms of Foc disease. In CR 50% affirmed that they could not
describe Foc, 26.6% gave a good description, 20% a general description and 3.3%
described the disease improperly. In NIC 90% declared that they could not describe the
Foc symptoms, 3.3% gave good descriptions to moderate or wrong description (Figure
24B). Afterward, when asked if they have Fusarium Wilt on the farm, 46.6% of CR
growers answered yes, 23.3% no and 30% did not know. In NIC 3.3% answered yes,
13.3% no and 83.3% did not know (Figure 25A). There was a significant difference
(p<0.0001) among the opinions concerning the presence of Foc disease at their farms.
33
In CR and NIC respectively 73.3% and 76.7% of the growers were able to recognize the
symptoms of disease on the farm. In 3.3% of CR farms the growers were not able to
recognize the disease while in NIC 16.7% of farms that had the disease was not
recognized by the growers (Figure 25B).
B
100
80
80
3.3
20
56.7
90
60
40
20
43.3
10
0
CR
Yes, I know Foc disease
No, I don't know Foc disease
Symptom descriprion (%)
Do you Know Foc disease (%)
A
100
3.3
3.3
3.3
26.6
60
90
40
50
20
0
NIC
CR
NIC
Could not describe
Good descripton
Moderate description
Wrong description
Figure 24: Interview results about ‘Fusarium Wilt knowledge’. (A) “Do you know Fusarium Wilt disease?” (B) “Could
you describe the Fusarium Wilt symptoms?” CR=Costa Rica. NIC=Nicaragua.
34
A
B
80
60
100
30
23.3
83.3
40
20
46.6
13.3
3.3
0
CR
Who Recognize Foc disease (%)
Do you have Foc disease (%)
100
80
3.3
6.7
16.7
16.7
3.3
3.3
73.3
76.7
CR
NIC
60
40
20
0
NIC
Yes
No
Do not know
Himself
Family
Employee
Nobody
Figure 25: Interview result about ‘Fusarium Wilt diagnoses’. (A) Do you have Fusarium Wilt diseased plants?” (B)
“Who perform the disease recognition?” CR=Costa Rica. NIC=Nicaragua.
In CR, growers stated that Foc disease arrived at their farms by: planting of
infested corms 13.3%, the usage of infested equipments (machete and boots) 10%, was
already present because land was used previously for commercial banana production
6.7%, did not know 70%. In NIC 100% of growers did not know anything concerning the
origin of Foc disease in their fields (Figure 26A). The most frequent method used to
control Foc disease in CR was “do not touch the diseased plant” with 36.6%, followed
by 20% who cut and chop the plant in small pieces after harvest, whereas 10% control
Foc based on seed selection and observation of signs of disease. Growers which have
no control methods in place in CR represented 33.3% of the growers. In NIC 93.3% of
growers had no methods of control in place, 3.3% cut and chop and 3.3% just grow a
new planting material to replace the lost one (Figure 26B). There were significant
differences (p<0.0001) between the control methods of Foc disease management.
35
B
100
80
80
60
70
100
40
20
6.7
10
Method of Control (%)
Foc Disease Origin (%)
A
100
33.3
60
36.6
40
20
10
20
13.3
0
0
CR
Seed
Tools
Old banana farm
Do not know
NIC
93.3
CR
3.3
3.3
NIC
Cut and chop
Seed selection and observation
Grown new seed
do not touch
No control in place
Figure 26: Interview results about ‘Fusarium Wilt dissemination and control’. (A) “Do you know how Fusarium Wilt
first arrived in your plantation?” (B) “How do you control Fusarium Wilt disease?” CR=Costa Rica, NIC=Nicaragua.
In CR and NIC the answers to the questions: 1) have you received training on
banana production and disease management 2) do you control Fusarium Wilt and 3)
how do you control Fusarium Wilt disease, were correlated with Foc incidence using
Contingence Tables and Correspondence Analyses Graphics. There was no significant
correlation of these questions with Foc incidence in CR. However in NIC, significant
differences were revealed concerning the fact as to whether growers had not been
trained in banana production (p=0.0068, r=0.45), do not control disease (p<0.0001,
r=0.65) and the method of control been ‘do not control’ (p<0.0001, r=0.78) as can be
observed by the Figure 27.
36
Figure 27: Nicaragua Correspondence Analyses for the questions: “Have you received training on banana production
and disease management?” “Do you control Fusarium Wilt?” “How do you control Fusarium Wilt?”
3.5.4. Seed Management
In CR 83.3% of growers produce their own banana seed by removing corms from
the same lot or farm, 13.3% received planting material from other farms and/or the own
farm and 3.3% get their planting material exclusively from other farms. In NIC all
growers generate their seeds from their own farm (Figure 28A). Concerning seed
treatment, in CR 23.3% do not practice any treatment on banana seed prior to planting,
46.6% perform only roots and pseudostem pruning, 13.3% perform the roots and
pseudostem pruning plus a natural seed treatment, other 13.3% perform the prunings
and in addition chemical seed treatment and the last 3.3% perform the prunings and
used to observe and select healthy plants prior to plant (Figure 28B).
37
A
B
100
3.3
13.3
80
60
40
83.3
100
20
Banana Seed Preparation (%)
Banana Seed Origin (%)
100
23.3
80
3.3
13.3
46.6
13.3
3.3
60
40
33.3
20
46.6
16.6
0
0
CR
NIC
Same farm
same farm and/or other farm
Other farms
CR
NIC
Cut root and topl
Cut root and top + natural treat.
Cut root and top + chemical treat.
Cut root and top + plant observation
Nothing
Figure 28: Interview result about ‘seed management’. (A) “From where do you obtain the banana seed used in your
farms?” (B) “What kind of seed preparation do you perform before it is grown?” CR=Costa Rica, NIC=Nicaragua.
Natural treat=treatment with ashes, calcarium, boiled water or sun expose. Chemical treat=nematicides.
3.6.
Discussion
After obtaining the data from the survey of growers in both regions, it was
possible to understand the importance that the banana crop represents for smallholders
in CR and NIC. Most of these farmers are traditional coffee producers and the usage of
banana became important at first only as shade for coffee, as is Erythrina poeppegiana
and the Cordia alliodora trees (Cerdán 2007). At the present time, banana are grown in
these intercropping systems but are now offering not just shade, but supplemental
income (Cerdán 2007). In NIC the fact that growers do not consider banana the most
important crop is associated with the fact that Yassica Sur and Monterrey are new
banana production zones as compared to the north Caribbean and south pacific coast
of NIC, and Turrialba in CR where Standard Fruit CO operated in the recent past.
Banana represents for the CR and NIC growers more than just a tool to produce
coffee it is now an important economic resource. Banana offers the growers additional
38
income and unlike coffee, this income is not received once a year but on a monthly
basis which represents a certain economic guarantee over time.
The amount of cultural management which banana receives would be related to
different aspects such as: crop importance and technical preparation obtained through
extension services in the case of NIC, and economic factors of the growers in both
countries. Fertilization management is an activity not fully implemented in both countries
regions. Less than 40% and not more than 3.3% of growers in CR and NIC respectively
apply fertilization products to banana. Cost of fertilizer products was an aspect
mentioned in the interviews. When fertilization was available it is usually used for coffee
and only secondarily on banana.
Often the products used to supply nutrients to crops are those generated on the
farm itself, such as: compost, chicken manure or those products available at low prices,
such as: K-mag and Urea. Despite the claim that basic cultural management of planting
material seed, flower and sucker pruning is used, the fields accessed in the study did
not demonstrate that those activities are regularly performed. Numerous pseudostem
per mat and small bunch quality were frequently observed. The reasons for this neglect
could be the lack of training as seen in the NIC example or simply the fact that the local
market and the puré industry do not require better quality fruit standards.
The information generated regarding their knowledge of Foc disease was very
interesting. Regardless of Foc incidence, the results generated showed that CR growers
are better informed about Fusarium Wilt and disease diagnosis when compared to NIC
growers. In CR 43% of growers knew Fusarium Wilt, and from the 50% of growers who
attempted to described the symptoms, 46% gave acceptable descriptions, commenting
in some cases not only on how it look, but how symptoms advance. In NIC 10% of
growers which stated to know the disease, only one third of it could properly explain the
plant symptoms The familiarity with the crop from the technical point of view is small in
Nicaragua. Approximately 90% of the growers could not discuss the disease. This could
be due to the fact that they have a low incidence of the disease.
In CR the result for the question “Do you have Foc diseased banana plants?”
resulted in 46% affirmative answers among growers, whereby the real number for Foc
occurrence was smaller. This indicates that symptoms of bacterial wilt caused by Moko
39
disease (Pseudomonas solanacearum) and Erwinia musae, present at these zones is
bring mis-identified as Fusarium Wilt. In all the zones studied the growers alone are
responsible for disease identification. There was no indication of the existence of
government extension services supporting disease diagnose and management. Foc
disease control, based on the strategy of cutting down and chopping up the pseudostem
into small fragments, was the most often performed management techniques used
among the growers in CR and NIC. However the level of control is not very good,
considering that dissemination is promoted by involuntary movement of those fragments
around and in the fields. In CR control of Foc is based on allowing the plants to die
without removal or chopping. Moore et al. (1999b) had reported the in situ control
method used in Australia against ‘race 4’, where herbicide applications are used here
too. In NIC control measures are seldom performed and this added to low levels of
training in banana and disease management were significantly correlated with Fusarium
Wilt incidence. This suggests that training must be improved to reduce disease levels.
Finally, it was shown that Foc clean in-vitro plants are not used by smallholders
in CR or NIC. The main source of seeds is their own farms and in some cases as
reported in CR seeds from neighbors. This is a main source of disease dissemination,
and will continue to distribute Foc disease in the region. Seed treatment in CR an NIC
could have a positive effect on control of the banana weevil (Cosmopolites sordidus)
and nematodes, because control is based in root pealing and nematicide application.
Effective seed treatment was revealed in 3.3% of the farms in both CR and NIC, based
on the plant observation at the moment of seed selection.
3.7.
Conclusion
(a)
Growers responses has revealed that their understanding of biological and
curative disease control is limited. However their broad knowledge of cultural
disease management practices could explain why they are able to produce
banana in the presence of the wilt disease problem on susceptible ‘Gros Michel’.
(b)
In the Nicaragua studied zones, the low rate of banana production and
disease control training seems to have a strong effect on Fusarium Wilt incidence
and could be a future constrain to ‘Gros Michel’ banana production if expanded.
40
3.8.
Recommendation
(a)
Develop training programs for growers concerned with banana production,
which include topics about crop management, disease diagnosis and control on
the main banana diseases and pests.
(b)
Develop strategies for Fusarium Wilt control that can be easily adhered to
by Costa Rican and Nicaraguan smallholders, based on the intellectual and not
economic input. Good agricultural practice such as selection and multiplication of
banana seeds would contribute to Fusarium Wilt incidence reduction.
41
Chapter 4 Investigation on the Pathogenicity of CR Isolates of Foc
CHAPTER 4
4.
4.1.
Investigation on the Pathogenicity of CR Isolates of Foc.
Introduction
The Fusarium spp. is commonly associated of crop plants causing disease in a
broad number of those that are good hosts (Ploetz 2005b, Pérez-Vicente 2010a).
Therefore, correct identification plays an important role in both survey work on the
occurrence of Fusarium Wilt and in greenhouse studies to determine the pathogenicity
of isolates.
Three different criteria are presently used for taxonomic classification of
Fusarium species. With morphological criteria, Fusarium spp. can be identified by
pigmentation promoted by growth on specific media and the size and shape of
reproductive structures. The vegetative compatibility groups (VCG) test is a biological
tool used for identification especially of those isolates casing wilt disease (PérezVicente 2010b). The usage of phylogenetic criteria for Fusarium diagnostics is based on
DNA polymorphism and genomic sequences which can be used to establish differences
and relationships among individuals and populations (Martínez et al. 2010). In addition
VCG techniques and phylogenetic criteria are expensive and require specific
equipments. In the presents studies morphological identification was the only
methodology available for selection of isolates considered to be Fusarium oxysporum.
After identification to species a pathogenicity test was performed in the greenhouse to
determine the virulence of the isolates.
4.2.
Experimental Site
The pathogenicity test was carried out in a greenhouse at the Tropical Agronomic
Center of Research and Teaching (CATIE) located in Turrialba, Costa Rica. The
isolates were cultured and stored in the Plant Protection Laboratory. The greenhouses
were located 602 meters above sea level and the daily temperature averaged 21,6oC
with a relative humidity of 87%.
42
4.3.
Material and Methods
The samples were processed within 24 hours of field sampling. The general
laboratory procedures followed were: isolation from plant tissue, strain purification,
identification and storage (Figure 29). The isolates were grown in Petri dishes, the
spores collected by washing and sieving, spore density adjusted by dilution and plants
inoculated (Figure 30).
Figure 29: Isolate preparation steps. (a) mycelial growth (b) isolate purification (c) morphological identification and (d)
isolate storage.
Figure 30: Pathogenicity preparation steps (a) spores separation; (b) spore sieving; (c) spore density adjustment and
(d) plant inoculation.
4.3.1. Plant Material
Micropropagated ‘Gros Michel’ (AAA) banana plantlets produced by CATIE
Biotechnology Laboratory with 9 months old and averaged 7.5cm in size were used to
conduct the pathogenicity test.
4.3.2. Isolate Preparation
By immersion, 5 to 6 pseudostem and 5 to 6 corm tissue segments with
0.5x0.5cm in size were separately sterilized for 3 minutes in a 2.5% sodium hypochlorite
solution (NaCl 2.5%) followed by three rinses in sterile distilled water of 3 minutes each
(Pocasangre et al. 2000). Excess water was then removed on sterilized paper towels
and 5 tissue segments were equally distributed on 100mm diameter Petri dishes
43
containing 10% Potato Dextrose Agar (10% PDA). Five replicate were prepared. The
dishes were incubated for 72hrs at 28oC to 30oC in the dark prior to colonization rate
calculation, as follows.
% =
° 5 ∗ 100
° Fungal colonies were purified by removing a very small segment of mycelium
from the colony edge. These were placed in the center of 100mm diameter Petri dishes
on full strength Potato Dextrose Agar (100% PDA). The dishes were incubated 6 to 7
days at 28 to 30oC in the dark.
The morphological identification was then performed under the microscope to the
genus Fusarium, using primary and secondary characters that were visually observable
(Leslie 2006). The collected data was noted on the “Fusarium Culture Identification
Form – FCI003” (Annex 03) for each isolate.
Spores were stored in Eppendorf and Cryobank tubes. The former tubes were
use to store isolates over shorter periods of time by dragging sterile filter paper slices
over the isolate mycelium. The strips were then placed in the tubes, sealed, labeled and
storage at 5oC. The Cryobank tubes were used for long term conservation. The
mycelium was removed from the Petri plate and placed inside the cryo preservative
tube, which were gently agitated to distribute the isolate homogeneously. The cryo
preservative liquid was removed prior to storage at -20oC.
4.3.3. Plant Inoculation
Spores were removed from the Petri plates in 25ml of sterile water and gently
scraping the media surface with a sterile plastic spreader with round corners. The
acquired solution was sieved through gauze and decanted into 250ml beakers. Spores
counting were carried out with the Neubauer Hemocytometer slide and the final solution
adjusted to 1.5x10-6 spores/ml by adding sterile distilled water (Chaves et al. 2009).
Banana plantlets were removed carefully from the tray and placed inside plastic
containers containing 300ml of spore solution at 1.5x10-6 spores/ml. The roots system
was submerged for 15 minutes under gentle agitation before being planted in 300ml
44
pots in sterile substrate that was composed of equal parts of sand and clay with a pH of
6.2 (Chaves and Pocasangre 2010). The plants were watered as needed.
4.3.4. Media Preparation
One liter of distillate water was warmed in a beaker on a hotplate for a few
minutes under a magnetic agitation. Standard amounts of Potato Dextrose Agar and
Agar were added to the beaker while maintaining the agitation until homogeny solution
was obtained. 200 ml of the media was then transfer to five, 250ml Erlenmeyer and
covered with aluminum paper prior to sterilization. The 10% PDA ingredients were: 1
liter distilled water; 3.9 grams of Potato Dextrose Agar (PDA); 20 grams of Agar. The
100% PDA ingredients were: 1 liter distilled water; 39 grams of Potato Dextrose Agar
(PDA); 5 grams of Agar.
4.3.5. Treatment Application
A total of 17 isolates were evaluated for disease incidence, severity and negative
effects on plant growth. All treatments except the control were inoculated with specific
isolates of Fusarium recovered from CR diseased plants during the survey.
Weekly evaluations were performed during the bioassay. The bioassay ended
when the majority of treatments and repetitions were severely damaged by Foc wilt
disease symptoms. Symptoms index included: stem base splitting, yellowing and wilt.
The plant growth variables were: plant height, plant diameter at stem base, leaf number
and leaf area index. Stem base splitting was graded by frequency and yellowing and wilt
were graded according INIBAP grades compiled by Orjeda, (1998). At the end of the
greenhouse evaluation, the roots were carefully washed removing the soil substrate.
Fresh roots, and shoot weight were measured and the vascular internal symptoms of
discoloration graded according INIBAP scales (Orjeda 1998).
Four randomly selected plants from each isolate were selected to establish a
causal relationship between tissue colonization of the Fusarium isolate and symptoms
of disease. The pseudostem and corm tissue segments in 3 repetitions were placed on
of 100 mm Petri plates on 100% PDA as described in 4.3.2. The colonization rate was
determined using the following formula.
45
% =
4.4.
° 3 ∗ 100
° Nicaraguan Isolates
In Nicaragua, 18 Fusarium isolates were recovered from diseased plants and
stored at the Phytopathology Laboratory of the National Autonomy University of
Nicaragua (UNAN) in León for pathogenicity testing. The tests were to be performed by
local staff under the same protocol used above. This decision was based on the
agreement sign in October 17th 2008 in El Salvador by 9 countries of Centro America
and Caribbean represented by “Organismo Internacional Regional de Sanidad
Agropecuaria” (OIRSA) and issued in San Salvador which prohibits the movement of
plants and microorganisms across borders to avoid dissemination of Foc strains and
due the menace of Foc Tropical Race 4 (OIRSA 2008).
4.5.
Statistics
One way analysis of variance (ANOVA) was performed for each set of data
separated. Fishers Least Significance Difference (LSD) was used to detect significant
differences between means (p<0.05).
Category analysis was performed for severity and growth variables. The severity
variables such as yellowing, wilt, pseudostem splitting, corm discoloration as well as the
growth variables of shoot height, stem diameter, leaf area index, leaf number and plant
weight values were transformed to 0–1 intervals. Those intervals were separated into
four pathogenic value categories as follows: category 1 (0 until 0.25), category 2 (0.26
until 0.50), category 3 (0.51 until 0.75) and category 4 (0.76 until 1). As result the
category analysis grouped the treatments with the 25% poorest results.
Pearson’s correlation coefficient was used to establish significant (p<0.05)
relationships of incidence with severity and growth effects. The statistical software
Infostat ® version 2009e was used to perform the analyses.
46
4.6.
Results
4.6.1. Morphological Characterization
In CR all 17 isolates had showed abundant mycelia with cottony appearance.
Isolates showed a variation in color, which ranged between the pink and purple
coloration (Figure 31). Macroconidia had 3 to 4 segments, were straight and not curved,
had mostly blunted apical cells and barely notched foot cells. Microconidia were
generally oval shaped not segmented. No chlamydospores were observed (Table 6).
In NIC all 18 isolates produced abundant mycelia with cottony appearance. The
color of the isolates also varied in color, between pink and purple (Figure 32).
Macroconidia had 3 segments, the dorsal sides were curved more than ventral side
shape, the apical cell was blunted and the foot cell was slightly notched. The
microconidia were generally oval shaped and not segmented. No chlamydospores were
observed (Table 7).
47
Foc1
Foc2
Foc4
Foc5
Foc6
Foc7
Foc8
Foc10
Foc11
Foc12
Foc13
Foc14
Foc15
Foc16
Foc17
Foc18
Foc19
Figure 31: Color variation of Fusarium isolates recovered from ‘Gros Michel’ (AAA) banana plants in Costa Rica.
Figure 32: Color Variation in Fusarium isolates recovered from ‘Gros Michel’ (AAA) banana plants in Nicaragua.
48
Table 6: Morphological characteristics of Fusarium isolates recovered from Costa Rican diseased banana.
Isolate
Color on PDA
FOC 1
Macroconidia
Microconidia
Shape
Apical cell
Foot cell
Segments
Shape
Segments
Pink light
Straight
Blunt
Barely Notched
3
Kidney Form
0
FOC 2
Pink light
Straight
Papillate
Barely Notched
4
Kidney Form
0
FOC 4
Purple
Straight
Hooked
Barely Notched
3
Oval
0
FOC 5
Purple
Straight
Papillate
Barely Notched
3
Oval
0
FOC 6
Reddish
Straight
Blunt
Barely Notched
3
Kidney Form
0
FOC 7
White
Dorsal Side curved/ventral Side
Blunt
Barely Notched
3
Kidney Form
0
FOC 8
Reddish
Straight
Hooked
Barely Notched
4
Oval
0
FOC 10
Purple dark
Straight
Blunt
Barely Notched
3
Kidney Form
0
FOC 11
Pink light
Tapering
Barely Notched
4
Oval
0
FOC 12
Pink light
Blunt
Foot Shaped
3
Kidney Form
0
FOC 13
Pink light
Straight
Blunt
Barely Notched
3
Oval
0
FOC 14
Pink light
Straight
Blunt
Barely Notched
3
Oval
0
FOC 15
White
Straight
Blunt
Barely Notched
3
Kidney Form
0
FOC 16
Purple light
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
FOC 17
Pink light
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
FOC 18
Pink light
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
FOC 19
Purple light
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
Table 7: Morphological characteristics of Fusarium isolates recovered from Nicaraguan diseased banana.
Isolate
Color on PDA
FOC 1
Macroconidia
Microconidia
Shape
Apical cell
Foot cell
Segments
Shape
Segments
Reddish
blunt
barely notched
3-4
eliptical
0
FOC 2
Pink
blunt
barely notched
3-4
kidney shaped
0
FOC 3
Pink light
blunt
barely notched
3-4
oval
0
FOC 4
Purple
papillate
barely notched
3-4
oval
0
FOC 5
Reddish
needle like
tapering
distinctly notched
3-4
oval
0
FOC 6
Purple
needle like
tapering
barely notched
eliptical
0
FOC 7
Pink light
blunt
barely notched
eliptical
0
FOC 8
Pink light
blunt
barely notched
oval
0
FOC 9
Pink
papillate
barely notched
0-3
oval
0
FOC 10
White
blunt
barely notched
3-4
oval
0
FOC 11
Purple light
blunt
barely notched
3-4
eliptical
0
FOC 12
Purple light
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
3-4
FOC 13
Reddish
n.a.
n.a.
n.a.
n.a.
n.a.
FOC 14
Pink light
needle like
papillate
barely notched
3-4
round
0
FOC 15
Purple light
needle like
blunt
barely notched
3-4
round
0
FOC 16
Pink light
papillate
barely notched
3
oval
0
FOC 17
FOC 18
Orange
Purple
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
4.6.2. Field Sampling and Vascular Tissue Colonization
Colonization of the pseudostem tissue segments by the isolates recovered from
CR was infected on the average of 62% by Foc, with infection ranging from 4 to 100%.
49
The corm segments had an average of 54% colonization and ranged from 0 to 100%. In
NIC the pseudostem segments showed 45% colonization with Foc and ranged between
20 to 76%. The corm segments were infected 89% with Foc and ranged from 44 to
100% (Figure 33A).
In CR 65% of Fusarium isolates were obtained from pseudostem tissue and 35%
from corm tissue. Conversely, in NIC 39% were from the pseudostem and 61% from
corm tissue (Figure 33B).
A total of 17 isolates were recovered in CR and 18 isolates in NIC. Isolates were
recovered from Gross Michel (AAA) diseased plants. The number of isolates varied
among the banana samples and localities ranging from 1 to 4 isolates per sample
100
A
100
80
60
40
20
62%
54%
45%
89%
CR stem
CR corm
NIC stem
NIC corm
0
Foc isolates origin distribution (%)
vascular tissue segment colonization (%)
(Table 8).
80
B
65%
39%
35%
61%
60
40
20
0
Costa Rica
Nicaragua
corm
stem
Figure 33: Fusarium colonization levels in Costa Rica and Nicaragua banana. (A) Frequency of pseudstem and corm
tissue colonization of Costa Rica and Nicaragua field samples. CR=Costa Rica, NIC=Nicaragua, Stem=Pseudstem.
(B) Share of recovered Fusarium spp. isolates by vascular tissue type in Costa Rica and Nicaragua field.
50
Table 8: Fusarium spp. isolates origin by vascular tissue type, cultivar, farm, zone and country.
Country and Zone
Farm
Cultivar
Stem tissue
Corm tissue
Total
Costa Rica - Zone1
Farm 7
Gros Michel (AAA)
2
2
4
Costa Rica - Zone1
Farm 8
Gros Michel (AAA)
1
1
2
Costa Rica - Zone1
Farm 10
Gros Michel (AAA)
1
0
1
Costa Rica - Zone1
Farm 13
Gros Michel (AAA)
2
0
2
Costa Rica - Zone1
Farm 14
Gros Michel (AAA)
0
1
1
Costa Rica - Zone2
Farm 17
Gros Michel (AAA)
1
1
2
Costa Rica - Zone2
Farm 19
Gros Michel (AAA)
2
0
2
Costa Rica - Zone2
Farm 27
Gros Michel (AAA)
1
1
2
Costa Rica - Zone2
Farm 30
Gros Michel (AAA)
1
0
1
11
6
17
Total
Nicaragua - Zone1
Farm 1
Gros Michel (AAA)
1
2
3
Nicaragua - Zone1
Farm 3
Gros Michel (AAA)
1
2
3
Nicaragua - Zone1
Farm 4
Gros Michel (AAA)
0
3
3
Nicaragua - Zone1
Farm 5
Gros Michel (AAA)
2
0
2
Nicaragua - Zone1
Farm 6
Gros Michel (AAA)
2
2
4
Nicaragua - Zone1
Farm 8
Gros Michel (AAA)
1
0
1
Nicaragua - Zone1
Farm 14
Gros Michel (AAA)
0
2
2
7
11
18
Total
4.6.3. Pathogenicity Test – Incidence and Severity
At the end of the 3rd week 82% of the isolates showed symptoms of Foc
expressed as leaf yellowing, wilting or stem base splitting. The pathogenic isolates
were: T1, T2, T5, T6, T7, T8, T11, T12, T13, T14, T15, T16, T17, T18 and T19. Until the
end of the 6th week 100% of the isolates showed typical Foc disease symptoms. At the
end of the bioassay only the control plants did not show any symptoms of Foc disease.
Results could be seen in Figure 34.
The Foc incidence differed significantly (p<0.0001) between the control and the
plants treated with the isolates. Lower disease incidence was found in T4, T6 and T10.
External severity differed significantly (p<0.0001) among treatments. Treatments T4, T6
and T10 were slightly similar to control with regards to external and internal symptoms.
T1, T5 and T12 had developed the most severe external symptoms and T11 and T16
caused the most severe internal symptoms Results could be seen in Table 9.
51
100
90
80
Incidence (%)
70
60
50
40
30
20
10
0
Zero
1st
2nd
3rd
4th
5th
6th
week
T0
T1
T2
T4
T5
T6
T7
T8
T10
T11
T12
T13
T14
T15
T16
T17
T18
T19
Figure 34: Effect of 17 Fusarium oxysporum f. sp. cubense isolated taken from the field on Wilt Disease incidence of
‘Gros Michel’ (AAA) banana plants over 6 weeks. n=10.
Table 9: Incidence and severity of 17 Fusarium oxysporum f. sp. cubense isolates in ‘Gros Michel’ banana after 6
weeks. n=10.
Treatment (Foc)
Incidence
T0 - Control
0a
T10
3b
T6
T4
External Symptom
Yellowing
Internal Symptom
Wilt
Stem Split
Discoloration
1a
1a
0a
1a
1a
1.3 ab
3b
1a
3b
1.2 a
1.1 ab
1 ab
1a
3b
1.2 a
1.1 ab
1 ab
1.4 a
T18
9c
1.6 ab
1.9 b
8 cd
3b
T15
9c
3.2 de
3.1 cd
8 cd
3.6 bcd
T13
9c
3.3 def
3.3 cde
T8
10 c
3.4 def
3.2 cde
10 d
8 cd
4.1 cdef
4.2 cdef
T2
10 c
3.3 def
3.3 cde
9d
3.7 bcde
T7
10 c
3.8 efg
3.9 defg
9d
4.6 ef
T5
10 c
4.6 g
4.5 g
6c
3.7 bcde
T19
10 c
2.3 bc
2.9 c
9d
3.4 bc
T12
10 c
4.5 g
4.5 g
10 d
4.4 def
T11
10 c
3.4 def
3.5 cdef
6c
5f
T1
10 c
4.3 g
4.2 fg
T17
10 c
2.9 cd
2.8 c
8 cd
T16
10 c
4.1 fg
4 efg
10 d
4.9 f
T14
10 c
3.8 efg
3.8 defg
10 d
4.5 def
10 d
4.5 def
3.6 bcd
Data regards 6th week evaluation without transformation. Different letters means significant differences (p<=0.05).
52
4.6.4. Pathogenicity Test – Growth Variables
Growth differed significantly among treatments. Treatments T4, T6 and T10 were
not significantly different from the control with regards to plant size, stem diameter, leaf
area index and leaf number. T5 and T11 revealed lower growth effect. The weight of the
shoot, roots as well as the total plant differed significantly (p<0.0001) among treatments.
Treatments T4, T6 and T10 reveal similar weight than the control T0 whereas T11 and
T16 showed significantly lower weights Results could be seen in Table 10.
Table 10: Effect of 17 Fusarium oxysporum f. sp. cubense isolates on plant growth of Gros Michel in-vitro plants over
6 weeks.
Treatment (Foc) Plant Size (cm)
Stem base
Leaf Area Index
Diameter (mm)
Leaf Number
Above Ground
Root Weight (gr)
Weight (gr)
Total Plant
Weight (gr)
T0 - Control
11.80 a
1.03 a
15.19 a
6.0 a
18.30 a
7.61 ab
25.91 a
T4
11.45 a
0.99 ab
12.95 ab
5.6 ab
16.30 ab
7.00 bc
23.30 ab
T10
10.40 ab
0.91 abc
15.06 a
5.9 a
14.65 b
6.01 c
20.66 b
T6
10.35 ab
0.92 abc
12.77 ab
5.5 ab
17.06 ab
8.18 a
25.24 a
T2
9.20 bc
0.86 bcd
7.80 cdef
5.4 abc
11.03 c
4.17 d
15.20 c
T8
9.20 bc
0.76 cdef
7.28 cdefg
5.2 abc
7.75 def
4.21 d
11.96 cd
T7
8.75 bc
0.80 cde
4.91 efghi
4.7 bc
7.24 def
4.03 d
11.27 de
T15
8.35 c
0.72 def
8.88 cd
5.0 abc
6.16 efgh
1.84 hi
8.00 efgh
T14
8.05 c
0.68 ef
5.69 defghi
4.6 bc
5.21 fgh
2.79 fgh
8.00 efgh
T19
8.05 c
0.69 def
5.4 abc
6.00 efgh
1.59 i
7.59 fgh
T12
7.90 c
0.61 fgh
4.41 fghi
4.5 bc
3.61 h
2.97 efg
6.58 gh
T16
7.60 cd
0.60 fgh
7.07 cdefgh
4.5 bc
4.13 gh
1.46 i
5.59 h
T18
7.50 cde
0.67 efg
4.6 bc
8.66 cde
2.23 ghi
T13
7.45 cde
0.63 efg
8.30 cde
4.3 c
6.69 efg
3.30 def
9.99 defg
T1
7.40 cde
0.66 efg
4.21 ghi
4.5 bc
6.07 efgh
1.81 hi
7.88 efgh
12.71 ab
10.53 bc
10.89 def
T17
7.40 cde
0.68 ef
9.88 bc
4.9 abc
5.78 fgh
1.60 i
T5
5.95 de
0.50 gh
3.74 hi
3.1 d
9.38 cd
3.96 de
13.34 cd
7.38 gh
T11
5.70 e
0.45 h
3.22 i
3.0 d
4.03 gh
2.36 fghi
6.39 h
Data regard 6th week evaluation without tranformation. Different letters means significant differences (p<=0.05).
4.6.5. Categories Analysis
Regarding external and internal symptoms, the treatments T12, T16, T1, T14,
T7, T5 and T8 were placed in category 4 and were found to have stronger virulence
than the other isolates. The isolates T6, T4 and T10 were placed in category 1 with the
weakest level of virulence (Table 11). The isolates T11, T5, T16, T12, T1 and T14 had
less effect on plant development and were placed in category 4 whereas T4, T6 and
T10 had a greater impact on plant growth and were thus ranked placed in category 1
53
(Table 12). Correlation analyses demonstrated a relation between incidence and
yellowing (p<0.0001, r=0.897), wilt (p<0.0001, r=0.92) and discoloration (p<0.0001,
r=0.842). In addition, the growth variables were also correlated with: above ground
shoot weight (p<0.0001, r=-0.798), root weight (p=0.001, r=-0.705), total weight
(p<0.0001, r=-0.782), shoot height (p=0.016, r=-0.557), pseudostem diameter (p=0.019,
r=-0.543), Leaf area index (p<0.0001, r=-0.85) and leaf number (p=0.006, r=-0.611)
(Table 13). The severity of symptoms and growth development caused by Foc isolates
in Category 1 and 2 can be seen in Figures 35 and 36.
Table 11: Categories of virulence, external and
Table 12: Categories of virulence, growth development.
internal symptoms of Fusarium Wilt
Results
transformed
(results<25%),
for
0–1
intervals.
C2=category2
Results
C1=category1
(results
25-50%),
transformed
for
0–1
intervals.
C1=category1
(results>75%), C2=category2 (results 50-75%), C3=category3
(results 25-50%) and C4=category 4 (results<25%).
C3=category3 (results 50 – 75%) and C4=category4
(results>75%).
Categories
C1
C2
C3
C4
Categories
Treatments (Foc)
Growth
T0
1.00
T4
0.88
T6
0.88
T10
0.82
T2
0.54
Treatments (Foc)
External & Internal
Symptoms
T0
0.01
T6
0.04
T4
0.06
T10
0.10
T18
0.45
T8
0.44
T19
0.63
T7
0.37
T15
0.69
T19
0.36
T17
0.70
T18
0.34
T2
0.75
T13
0.76
T15
0.32
T11
0.78
T13
0.29
T8
0.80
T17
0.28
T5
0.86
T14
0.26
T7
0.89
T1
0.20
T14
0.90
T12
0.19
T1
0.92
T16
0.97
T16
0.18
T12
1.00
T5
0.17
T11
0.01
C1
C2
C3
C4
54
Figure 35: Category 1 of incidence, severity and growth. Represented by control treatment T0 and inoculated
treatments T6, T4 and T10.
Figure 36: Category 4 of incidence, severity and growth. Represented by inoculated treatments T12, T16, T1, T14,
T17, T5, T8, T11 and T13.
55
Table 13: Statistical correlation between incidence, severity and growth variables of pathogenicity test treatments.
Pearson significance of 0.05.
growth variables
severity
Variables
Incidencia
(p)
( r)
Discoloration
0.0000005
0.8971
Yellowing
0.0000001
0.9202
Wilt
0.0000114
0.8426
Above ground weight
0.0000701
-0.7989
Root weight
0.0010733
-0.7055
Total weight
0.0001263
-0.7820
Plant Size
0.0162620
-0.5573
Diameter
0.0196209
-0.5439
Leaf area Index
0.0000079
-0.8502
Leaf number
0.0069603
-0.6119
4.6.6. Segment Colonization
The vascular tissue colonization of pseudostem and corm of all the isolates
tested confirmed the relationship between the presence of Foc and wilt disease
development. The vascular tissue segments from the control plants were completely
free of fungal infection.
4.7.
Discussion
Growth of the isolates on PDA resulted in typical Foc pigmentation, as mentioned
by Burgess et al. (1989), Summerell et al. (2003), Leslie and Summerell (2006) and
Pérez-Vicente (2010). The conidia formed were typical for Foc and absence of
chlamydospores in PDA was previously mentioned by Burgess et al. (1994) and Leslie
and Summerell (2006). The Light pink and purple coloration was previously observed on
PDA by other researchers (Rajapakse et al. 2005, Pérez-Vicente 2010).
The field recovered isolates and bioassay pathogenicity tests demonstrate that
the Foc isolates found in banana tissue in CR and NIC are able to re-colonize banana
vascular tissue especially the corm and peudostem. The corm is the main nutrient
reserve of the plant (Stover 1962, Goés and Moretto 1997) and the fungus uses
possibly as a nutrient source (Pocasangre 2000).
56
All isolates infected ‘Gros Michel’ banana with different levels of virulence
detected. Foc symptom development was observed in 82% of inoculated plants after 3
weeks and in 100% of the plants after 6 weeks. The symptoms observed in the
greenhouse tests included leaf yellowing, wilting and stem base splitting. Similar results
were obtained for Foc race1 where external symptoms appeared between the 3rd and
4th week under controlled conditions (Stover 1959, Hadi and Ghorab 1987, Sivamani
and Gnanamanickam 1988). Vinh et al. (2001) demonstrate that Foc race 1 was
capable of infecting the variety Chuoi (AAA) on the 6th week after inoculation and
experiments performed by Sabadel and Hernández (2003) and Saravanan et al (2007)
demonstrate that external symptoms became visible only after the 12th week. Similar
results were obtained in the present experiments with isolates T4, T6 and T10 which
showed late external symptom expression.
Lower external and internal symptom development as well as increased plant
growth rates were found for the isolates T4, T6 and T10 when compared to the other
isolates. These isolates were actually very similar to that obtained in the control
treatment and may indicate that these were pathogenic with lower virulence or even
non-pathogens. For this reason they were placed into category 1 with the control.
Variation in results among the isolates were expected due differences in fungal
virulence. Similar results were obtained by Vinh et al. (2001), Kung’u and Jeffries
(2001), Kumar et al. (2006) and Groenewald et al. (2006). The results obtained with the
Foc isolates T4, T6 and T10 suggest the possibility of using these good isolates that are
good colonizers for bio-control. The extensive endophitic growth capability and lack of
disease expression may lead to cross protection effects (Sikora et al. 2008). Nel at al.
(2006), Forsyth et al. (2006), showed reduction on Foc race 4 in ‘cavendish’ banana
plant by the usage of non-pathogenic Fusarium oxysporum strains. The modes of action
was described by Fravel et al. (2003) as direct antagonism of the non-pathogenic strain
to the pathogen and/or indirect antagonism mediate through the host plant. Increased
growth
in
banana
that
was
colonized
by
non-pathogenic
Foc
endophytic
microorganisms also has been obtained by Ting et al. (2008). Chaves et al. (2009) had
reported roots weight gains of 58% over the non treated control when compared with
endophytic fungis and bacterias combined treatment.
57
A positive correlation (p<0.05) existed between incidence and severity and a
negative correlation with growth parameters. The isolates with high incidence and
severity caused significant growth reductions.
4.8.
Conclusion
(a)
17 isolates were recovered from wilt diseased ‘Gros Michel’ in Costa Rica
and 18 isolates recovered in Nicaragua.
(b)
The pathogenicity tests demonstrated that all isolates recovered from
diseased banana tissue from Costa Rica were the Fusarium oxysporum f. sp.
cubense and that they had significant levels of virulence differences.
(c)
The small virulence demonstrated by treatment T4, T6 and T10 suggest
that they could be potential endophytic candidate for biological control to reduce
colonization of Wilt pathogen in banana.
4.9
Recommendation
(a)
Perform VCG tests on the 17 isolates from CR and 18 isolates from NIC to
obtain a better understanding regarding the epidemiology in those countries.
(b)
Evaluate the biocontrol effects by using the Treatments T4, T6 and T10
against identified strains of Fusarium oxysporum f. sp. cubense in ‘Gros Michel’
(AAA) plants.
(c)
Evaluate the treatments T4, T6 and T10 as endophytic growth promoter
microorganisms.
58
Paulo dos Santos Faria Lichtemberg M.Sc. Thesis
5.
References
Abdel-Hadi, M.A., and Ghorab, A.I. 1987. Studies on the root-lesion nematode
Pratylenchus penetrans (Cobb, 1917) Chitwood & Oteifa, in cloves. In:
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68
6.
6.1.
Annexes
Annex 1 – Field Form (FF001)
FIELD FORM
FF001
BLOCK 01 - Farm Identification
Farm Code
Grower Name
Date&Time
Farm Size (ha)
Asociated Size
FOC in farm
BLOCK 02 - Plot Mapping
Coffee Banana Trees Fields
Plot 1
Plot 2
Gros Michel/Coco
Gros Michel/Coco
Congo
Congo
Platano
Platano
Others
Others
Total
Total
BLOCK 03 - Plot Data
Plot 01
Plant Population
Count (un)
Density (ha)
Plot 02
Incidence (%)
Count (un)
Density (ha)
In Farm
Incidence (%)
Density (ha)
Incidence (ha)
Banana Plants
Coffee Plants
Trees
Stem Average P1
FOC BANANAS
Stem Average P2
Banana Observation
(P1&P2)
Trees Observation
(P1&P2)
Latitud
GPS DATA
Longitud
Altitude (m)
Acurance (m)
waypoint
Inclination
Plot 1
Plot 2
BLOCK 04 - Sample Identification
P1
PLOTS
SEVERITY
Plant 1
Plant 2
P2
Plant 3
Plant 1
Plant 2
Sample Code:
Plant 3
Farm Average
Plant Size
Plant Diameter
Leaf Number
Non-Func. Leaf
EXT. Yellowing
EXT. Wilt
LY leaf yellowing
PBS stem split
NLC leaf change
PC petiole colap
INTERNAL
Discoloration
Root Weight (g)
Functional (g)
Non Functional g
Necrosis Rate
69
6.2.
Annex 2 – Questionnaire
Improving small farm production and marketing of banana
under trees: Resource partitioning, living soils, cultivar
choice and marketing strategies.
2009
Interview No:
Date:
Grower:
Age:
Farm:
Size:
Address:
City:
Universidad de Bonn & Bioversity International
70
Información General para el Entrevistado, Introducción y Propósito de la Encuesta:
A través de esta encuesta deseamos buscar informaciones para el entendimiento de
los sistemas de producción y de las relaciones de trabajo de las fincas. Las
informaciones brindadas por los productores se mantendrán en anonimato para
salvaguardar su opinión. Estos resultados regresaran a su conocimiento través las
instituciones locales de extensión.
Actualmente se está comenzando el proyecto “Mejorando la producción y mercadeo
de bananos en cafetales con árboles de pequeños productores: Utilización de los
recursos, salud de los suelos, selección de cultivares y estrategias de mercado” en
Costa Rica. Este proyecto lo está implementando la institución Bioversity International
con el apoyo de instituciones locales de Costa Rica: Universidad de Costa Rica (UCR),
Centro Agronómico Tropical de Investigación y Enseñanza (CATIE) y Asociación de los
Productores Orgánicos de Turrialba (APOT).
Como parte del proyecto, estarán los estudiantes de maestría Christian Dold de
Alemania y Paulo Lichtemberg de Brasil realizando sus trabajos de grado en su
comunidad. Estos estudiantes tienen como objetivo principal entender las interacciones
entre árboles, banano y café en sistemas mixtos, evaluando: los efectos del sombreo
entre plantas y su crecimiento, productividad y salud.
Cristian y Paulo visitaran las fincas en dos etapa; en la primera, se hará una
caracterización de la propiedad a través de una encuesta y de mediciones en campo;
en la segunda etapa regresaran para hacer un estudio más detallado en las parcelas
establecidas dentro de las fincas en las mismas parcelas.
Las informaciones colectadas tendrán un análisis para el entendimiento de estas
interacciones y después serán compartidas con las instituciones locales para hacerlo
llegar al conocimiento de los productores. Agradezco desde ya la oportunidad que nos
da a tener este.
71
(1) Farm Section:
1. Could you make a general farm description? Telling what are the
activities here performed and what is subsistence and what goes to
commercialization?
2. There are other incomes in your family?
3. Which crop do you consider the most important? Why?
4. Why banana crop is important?
(2) Coffee Section
1. What amount correspond your coffee production?
2. How do you sell the coffee production?
3. Do you perform fertilization management in coffee?
4. How do you control disease and pests in coffee?
(3) Banana section
1. Have you received training on banana production and disease
management? Who provide? Could you tell me what did you learned?
2. How much banana your farm produce?
3. How do you sell the banana production?
4. Do you perform fertilization management in banana?
5. What type of fertilizers do you apply in banana?
6. Which crop management do you perform in banana?
(4) Banana Disease section
1. Which disease and plagues are the most common in your banana crop?
2. Who identify the disease and plagues in your farm?
3. Do you know Fusarium Wilt? What do you know about?
4. Do you have Fusarium Wilt diseased plants in your farm?
5. Do you know how Fusarium Wilt first arrived in your plantation?
6. How Fusarium Wilt diseased plants looks? Could you explain to me how
do you identify one Fusarium Wilt diseased plant?
7. What types of banana are most frequently affected by Fusarium Wilt?
8. How do you control Fusarium Wilt?
72
(5) Banana Seed Management section
1. Do you renovate the banana plants? How often?
2. From where do you obtain the banana seed used in your farm?
3. What seed type is your favorite? How do you select in field?
4. Could you explain to me what kind of seed preparation do you perform
before it is grown?
73
6.3.
Annex 3 – Fusarium Culture Identification (FCI003)
74
7.
Curriculum Vitae
Name:
Paulo dos Santos Faria Lichtemberg.
Place Date of birth:
Porto Alegre, August 8 , 1979.
Nationality:
Brazilian.
Civil Status:
Single.
Address:
Avenida Atlantica 4040, apt 303, B. Camboriu, Brazil, 88330027
th
(C/o Vera S F C Pereira).
Telephone:
(55) 48 3233 5931 (55) 47 3367 1903
E-mail:
[email protected]
Institution:
Date:
Degree to be obtained:
Supervisor:
Description:
Institution:
Date:
Degree obtained:
Supervisor:
Description
Department of Phytopathology & Nematology in Soil Ecosystems,
INRES, University of Bonn, Germany.
From 2008 until 2010.
Master in Science (M.Sc.).
Professor Dr. Richard A. Sikora.
Occurrence, incidence and grower perception of Fusarium oxysporum
f.sp. cubense in banana intercropped with coffee and trees in Costa Rica
and Nicaragua smallholders
Fundação Getúlio Vargas (FGV), Santa Cruz do Sul, Brazil.
From 2004 until 2005.
Master of Business Administration (MBA).
Dr. David Lobato.
Thesis work in Virtual Business Planning – Budget forecast for three
consecutive periods based on external influences.
Date:
Degree obtained:
Supervisor:
Department of Rural Engineer, Universidade Federal de Santa
Catarina, Florianópolis, Brazil.
From 1997 until 2002
Bachelor in Agronomy (B.Sc.).
Professor Dr. Antonio A. A. Pereira.
Description
Organic Bananas in Costa Rica. South Brazil Potentiality.
Institution:
Language Skills:
Portuguese
Mother tongue
English
Advanced
Spanish
Advanced
German
Beginner
Professional Experience:
Institution
Date
Position
Description
Philip Morris Brazil, Santa Cruz do Sul, Brazil.
January 2003 – August 2007
Leaf Blender Supervisor
• Maintenance of portfolio blend formulas;
• Development of blend formulas for new brands and family extension;
• IPM of stored products program coordinator;
75
Publications:
Lichtemberg, P.S.F., Pocasangre, L.E., Staver, C., Sikora, R.A. Current Status of Fusarium Wilt
(Fusarium oxysporum f. sp. cubense) in banana Gros Michel (AAA) at smallholder level in Costa Rica.
In: Reunión de ACORBAT (November 2010), Medellin, Colombia.
Pocasangre, L.E., Lichtemberg, P.S.F. 2010. Reconocimiento de la situación del Mal de Panamá en
América Latina y El Caribe. In: Taller de entrenamiento sobre el diagnóstico y characterización de la
marchitez por Fusarium o Mal de Panamá (January 2010), Turrialba, Costa Rica, 173p. Abstracts.
Comin, J.J., Lichtemberg, P.S.F., Sena, C. Miller, P.R.M., Pereira, A.A.A., Inácio, C.T., Lovato, P.E.,
Coan, L.F.B. 2002. Recuperação de áreas degradadas da Praia Mole. In: IV Congresso Brasileiro de
Sistemas Agroflorestais (October 2002), Ilhéus, BA, Brasil. Abstracts.
Lichtemberg, L.A., Schimitt, A.T., Malburg, J.L., Hinz, R.H., Lichtemberg, P.S.F. 1998. Effect of
bunch cover and chemical sprays on mechanical climatic and pest damage in Cavendish bananas. In:
Reunión de ACORBAT (November 1998), Guayaquil, Ecuador. Abstracts.
Lichtemberg, L.A., Schimitt, A.T., Malburg, J.L., Hinz, R.H., Lichtemberg, P.S.F., Stuker, H. 1998.
Effect of bunch cover under the bananas quality and production component. In: XV Congresso
Brasileiro de Fruticultura, Poços de Caldas, MG, Brasil. Abstracts.
Lichtemberg, P.S.F., Pocasangre, Dold, C., L.E., Staver, C., Sikora, R.A. Fusarium Wilt (Fusarium
oxysporum f. sp. cubense) in Gros Michel (AAA) bananas, the incidence at smallholder level in
Nicaragua. In:TROPENTAG 2010 (August 2010), Zurich, Switzerland. (Submitted for approval).
Lichtemberg, L.A., Sonego, M., Moreto, A., Lichtemberg, P.S.F. 2010. Cold damage in banana fruits
in Santa Catarina State, Brazil. In: International Horticulture Congress (August 2010), Lisbon,
Portugal. (Submitted for approval).
Lichtemberg, L.A., Malburg, J.L., Sonego, M., Moreto, A., Lichtemberg, P.S.F. 2010. Chilling damage
in banana leaves in South of Brazil. In: International Horticulture Congress (August 2010), Lisbon,
Portugal. (Submitted for approval).
Lichtemberg, L.A., Miranda, M., Sáes, L.A., Schwarz, S., Lichtemberg, P.S.F. Sonego, M., Damatto
Júnior, E.R., Negreiros, R.J.Z., Paulo, B.K., 2010. Banana sub-tropical production in Brazil. In:
International Horticulture Congress (August 2010), Lisbon, Portugal. (Submitted for approval).
References:
Dr. Richard A. Sikora Institute of Crop Science and Resource Conservation (INRES) Plant Pathology
Department: University of Bonn. Nussalle 9, Bonn, Germany ([email protected]) +49 228 732439.
Dr. Luis E. Pocasangre Regional Coordinator for Latin America and the Caribbean of the Program
for Commodities and Livelihoods Improvement. Bioversity International, CATIE, Turrialba, Costa Rica
([email protected]) +506 2556 4386.
th
Luciano Sehnem Product Development Asia, Philip Morris International (PMI). 16 floor, Menara
Milenium, Kuala Lumpur, Malaysia. ([email protected]) +60 (3) 2080 5635.
Dr. Paul Richard M. Miller Agricultural Science Center (CCA): Federal University of Santa Catarina
State (UFSC). Rodovia Ademar Gonzaga 1346, Florianopolis, Brazil. ([email protected]) +55 48
3721 5345
76

master thesis work 2010 - Sociedade Brasileira de Fruticultura

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