ࡱ > ~ 5 bjbjr2r2 XhXhݘ 2 ` ` ` ` ` t t t 8 $ t d \ 6 P p | d d d d d d d $ ^g j 9d ] ` @ P 9d ` ` h d ( ( ( ^ ` ` d ( d ( ( N Z [_ wT O\ 0 c d 0 d \ j d! * j ` [_ j ` [_ ( 9d 9d % F d j X & : Standard Operating Procedure (SOP) for screening for resistance to Black Sigatoka (P. fijiensis) in Bananas
Authors & Contributors
N. Kinalwa, C. Serebe, G. Mwanje, R. Kanaabi, J. Kisitu, M. Mulongo, G.V. Nakato*
*Correspondence: HYPERLINK "mailto:V.Nakato@cgiar.org" V.Nakato@cgiar.org
1. Introduction
Banana, and especially the cooking type known as East Africa highland banana, is a vital staple food and income crop for over 80 million people in East Africa. However, productivity has been declining, partly due to diseases such as Sigatoka leaf diseases. Sigatoka disease of banana is caused by fungi of the genus Pseudocercospora (previously called Mycosphaerella). Species associated with Sigatoka disease include; P. fijiensis, causal agent of black Sigatoka, P. musicola of yellow Sigatoka ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1094/PD-90-0684A","ISSN":"0191-2917","PMID":"30781160","abstract":"Black Sigatoka, also known as black leaf streak, is caused by Mycosphaerella fijiensis Morelet (anamorph Pseudocercospora fijiensis (Morelet) Deighton). It is the most significant disease of bananas and plantains (Musa spp.) because most of the economically important cultivars of exported and staple commodities are highly susceptible. The Caribbean is one of the few regions of the world where black Sigatoka is not widespread. Black Sigatoka has been reported in the Bahamas, Cuba, Hispaniola, and Jamaica (2). Yellow Sigatoka, caused by M. musicola Leach (anamorph P. musae (Zimm.) Deighton), has been recognized in Puerto Rico since 1938-1939 (3). In August 2004, symptoms resembling black Sigatoka were first observed in Aasco, Puerto Rico by extension personnel from the University of Puerto Rico. Since black and yellow Sigatoka produce similar disease symptoms, a survey was conducted in the western banana- and plantain-production region of Puerto Rico to confirm the presence of black Sigatoka. Leaf samples were collected from production fields near the towns of Las Marias, Maricao, and Aasco. Single-ascospore isolates were recovered using the discharge technique from moistened pseudothecia in necrotic lesions that were inverted over water agar, and ascospores were transferred to potato dextrose agar. The isolates were subcultured in potato dextrose broth for mycelium production. DNA was isolated from mycelium with the FastDNA kit (Q-Biogen, Irvine, CA) for 19 isolates. Internal transcribed spacer as well as the 5.8s rDNA regions were polymerase chain reaction amplified with primers specific to M. fijiensis or M. musicola (1). Amplification products (1,100 bp) were observed for 18 of the 19 isolates, 6 of which were M. fijiensis and the remaining 12 were M. musicola, while the positive controls for both species were also amplified with the respective primer pairs. M. fijiensis was recovered from production fields close to all three towns. The source of M. fijiensis in Puerto Rico is unclear, but it may have originated from introduced leaf material and/or wind dispersed ascospores from neighboring countries. The presence of black Sigatoka in Puerto Rico will most likely increase production costs where fungicide applications will be needed to maintain yields. The USDA-ARS, Tropical Agriculture Research Station is the official Musa spp. germplasm repository for the National Plant Germplasm System. As such, efforts are underway to introduce and evaluate blac","author":[{"dropping-particle":"","family":"Irish","given":"B M","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Goenaga","given":"R","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ploetz","given":"R C","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Plant disease","id":"ITEM-1","issue":"5","issued":{"date-parts":[["2006","5"]]},"page":"684","publisher":"Scientific Societies","title":"Mycosphaerella fijiensis, Causal Agent of Black Sigatoka of Musa spp. Found in Puerto Rico and Identified by Polymerase Chain Reaction.","type":"article-journal","volume":"90"},"uris":["http://www.mendeley.com/documents/?uuid=5fbce042-a457-38dc-bdc7-66b58ffca1dd"]}],"mendeley":{"formattedCitation":"(Irish et al., 2006)","plainTextFormattedCitation":"(Irish et al., 2006)","previouslyFormattedCitation":"(Irish et al., 2006)"},"properties":{"noteIndex":0},"schema":"https://github.com/citation-style-language/schema/raw/master/csl-citation.json"}(Irish et al., 2006) and P. eumusae of Eumusae leaf spot ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1094/PHYTO.2000.90.8.884","ISSN":"0031949X","abstract":"A previously undescribed leaf spot disease of banana has been discovered in southern and Southeast Asia. The fungus identified as the causal agent of this leaf spot has a Mycosphaerella teleomorph stage and a Septoria anamorph stage. Isolation and reinoculation of the fungus to banana reproduced symptoms and confirmed its pathogenicity. Phylogenic analysis based on sequences of the internal transcribed spacer and 5.8S ribosomal DNA regions from the different leaf spot pathogens of bananas was consistent with the definition of a new species. M. eumusae (anamorph S. eumusae) is the name proposed for the causal agent and Septoria leaf spot as the name for the disease. The presence of the pathogen has been confirmed in leaf specimens from southern India, Sri Lanka, Thailand, Malaysia, Vietnam, Mauritius, and Nigeria.","author":[{"dropping-particle":"","family":"Carlier","given":"Jean","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Zapater","given":"Marie Franoise","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Lapeyre","given":"Fabienne","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Jones","given":"David R.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Mourichon","given":"Xavier","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Phytopathology","id":"ITEM-1","issue":"8","issued":{"date-parts":[["2000"]]},"page":"884-890","publisher":"American Phytopathological Society","title":"Septoria leaf spot of banana: A newly discovered disease caused (anamorph Septoria eumusae) by Mycosphaerella eumusae","type":"article-journal","volume":"90"},"uris":["http://www.mendeley.com/documents/?uuid=6da30f0e-f7b1-3ce0-886f-aa60d9a17473"]}],"mendeley":{"formattedCitation":"(Carlier et al., 2000)","manualFormatting":"(Carlier et al., 2000","plainTextFormattedCitation":"(Carlier et al., 2000)","previouslyFormattedCitation":"(Carlier et al., 2000)"},"properties":{"noteIndex":0},"schema":"https://github.com/citation-style-language/schema/raw/master/csl-citation.json"}(Carlier et al., 2000; ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1371/journal.pgen.1005904","ISSN":"15537404","PMID":"27513322","abstract":"The Sigatoka disease complex, caused by the closely-related Dothideomycete fungi Pseudocercospora musae (yellow sigatoka), Pseudocercospora eumusae (eumusae leaf spot), and Pseudocercospora fijiensis (black sigatoka), is currently the most devastating disease on banana worldwide. The three species emerged on bananas from a recent common ancestor and show clear differences in virulence, with P. eumusae and P. fijiensis considered the most aggressive. In order to understand the genomic modifications associated with shifts in the species virulence spectra after speciation, and to identify their pathogenic core that can be exploited in disease management programs, we have sequenced and analyzed the genomes of P. eumusae and P. musae and compared them with the available genome sequence of P. fijiensis. Comparative analysis of genome architectures revealed significant differences in genome size, mainly due to different rates of LTR retrotransposon proliferation. Still, gene counts remained relatively equal and in the range of other Dothideomycetes. Phylogenetic reconstruction based on a set of 46 conserved single-copy genes strongly supported an earlier evolutionary radiation of P. fijiensis from P. musae and P. eumusae. However, pairwise analyses of gene content indicated that the more virulent P. eumusae and P. fijiensis share complementary patterns of expansions and contractions in core gene families related to metabolism and enzymatic degradation of plant cell walls, suggesting that the evolution of virulence in these two pathogens has, to some extent, been facilitated by convergent changes in metabolic pathways associated with nutrient acquisition and assimilation. In spite of their common ancestry and shared host-specificity, the three species retain fairly dissimilar repertoires of effector proteins, suggesting that they likely evolved different strategies for manipulating the host immune system. Finally, 234 gene families, including seven putative effectors, were exclusively present in the three Sigatoka species, and could thus be related to adaptation to the banana host.","author":[{"dropping-particle":"","family":"Chang","given":"Ti Cheng","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Salvucci","given":"Anthony","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Crous","given":"Pedro W.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stergiopoulos","given":"Ioannis","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"PLoS Genetics","id":"ITEM-1","issue":"8","issued":{"date-parts":[["2016","8","1"]]},"publisher":"Public Library of Science","title":"Comparative Genomics of the Sigatoka Disease Complex on Banana Suggests a Link between Parallel Evolutionary Changes in Pseudocercospora fijiensis and Pseudocercospora eumusae and Increased Virulence on the Banana Host","type":"article-journal","volume":"12"},"uris":["http://www.mendeley.com/documents/?uuid=3d1d1f88-ea3a-312d-82bd-7e47def4f084"]}],"mendeley":{"formattedCitation":"(Chang et al., 2016)","manualFormatting":"Chang et al., 2016)","plainTextFormattedCitation":"(Chang et al., 2016)","previouslyFormattedCitation":"(Chang et al., 2016)"},"properties":{"noteIndex":0},"schema":"https://github.com/citation-style-language/schema/raw/master/csl-citation.json"}Chang et al., 2016). Yellow Sigatoka symptoms are characterised by oval to round necrotic lesions, which first appear pale yellow on the lower surface of the leaf, and this differentiates it from black Sigatoka at early stages of lesion development. Damage is manifested as necrotic leaf lesions that reduce the functional leaf area and photosynthetic capacity, resulting in reduced crop yield and fruit quality leading to banana yield losses ranging from 20-50% ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1094/PHYTO.2000.90.8.884","ISSN":"0031949X","abstract":"A previously undescribed leaf spot disease of banana has been discovered in southern and Southeast Asia. The fungus identified as the causal agent of this leaf spot has a Mycosphaerella teleomorph stage and a Septoria anamorph stage. Isolation and reinoculation of the fungus to banana reproduced symptoms and confirmed its pathogenicity. Phylogenic analysis based on sequences of the internal transcribed spacer and 5.8S ribosomal DNA regions from the different leaf spot pathogens of bananas was consistent with the definition of a new species. M. eumusae (anamorph S. eumusae) is the name proposed for the causal agent and Septoria leaf spot as the name for the disease. The presence of the pathogen has been confirmed in leaf specimens from southern India, Sri Lanka, Thailand, Malaysia, Vietnam, Mauritius, and Nigeria.","author":[{"dropping-particle":"","family":"Carlier","given":"Jean","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Zapater","given":"Marie Franoise","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Lapeyre","given":"Fabienne","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Jones","given":"David R.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Mourichon","given":"Xavier","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Phytopathology","id":"ITEM-1","issue":"8","issued":{"date-parts":[["2000"]]},"page":"884-890","publisher":"American Phytopathological Society","title":"Septoria leaf spot of banana: A newly discovered disease caused (anamorph Septoria eumusae) by Mycosphaerella eumusae","type":"article-journal","volume":"90"},"uris":["http://www.mendeley.com/documents/?uuid=6da30f0e-f7b1-3ce0-886f-aa60d9a17473"]}],"mendeley":{"formattedCitation":"(Carlier et al., 2000)","plainTextFormattedCitation":"(Carlier et al., 2000)","previouslyFormattedCitation":"(Carlier et al., 2000)"},"properties":{"noteIndex":0},"schema":"https://github.com/citation-style-language/schema/raw/master/csl-citation.json"}(Carlier et al., 2000). Use of resistant cultivars is the most effective and sustainable solution to manage sigatoka. Therefore, screening of banana genotypes for resistance to black sigatoka is crucial for breeding programs to identify and develop resistant cultivars of bananas.
2. Purpose
The purpose of this SOP is to provide guidance to the breeding program of bananas in assessment of genetic resistance to black sigatoka disease.
3. Scope
This document contains the procedures to be followed when screening for resistance to black Sigatoka disease in bananas. It covers steps from isolating the pathogen from plant material, culturing of pathogen in the laboratory, experimental setup and management, data collection and curation.
4. Definition of terms
Hybrids: Varieties of banana plants generated after crossing two different varieties
Checks: Known Varieties susceptible or resistant to the constraint
Controls: These are the landraces of banana plants commonly cultivated for food in Uganda
5. Roles and Responsibilities
Research Technicians are responsible for Tissue Culture Lab plantlet generation, Inoculum preparation, Inoculation, Data collection, Data curation and Analysis.
Pathologists/Research Assistants is/are responsible for experiment Planning and Supervision.
Pathologist/Breeder are responsible for Data Analysis and publications.
Field Assistants are responsible for Data collection.
6. Procedure/Protocols
Step 1: Experimental Planning (Pathologist/ Research Assistant)
Materials and methods:
Planting materials:
Test genotypes and newly developed hybrids
Susceptible checks
Mbwazirume
Landraces
TM-28 OBINO LEWAI
Mchare
Resistant check
Calcutta 4
Other Materials include;
Sterile Loam soil, 13-liter plastic pots/poly-planters, Watering cans, Polysheets, black Sigatoka cultures
Step 2: Generating Plants for screening (Research Technician Tissue Culture Lab)
The genotypes to be screened are generated from Tissue culture (TC) Laboratory, then transferred to the humid chamber for 4 weeks for weaning, then hardened under shade for 4 weeks in the nursery/screen house until ready for open space planting, (refer to Weaning SOP IITA-BP-SOP06-06)
Step 3: Developing an Experimental design and layout (Research Assistant)
NB: Any design like CRD, RCBD etc., can be applied depending on prevailing conditions
In this case, a partially replicated experimental design (P-Rep) is developed using updated CycDesigN Computer software. This design is very useful when running experiments in batches in cases where plant genotypes are many and can`t be evaluated at once, and availability of space is a limiting factor. It can allow running repeated trail evaluations at different locations and time.
3.1 CycDesigN (Illustration)
Below is a layout of a P-rep design for 51 parental genotypes including checks developed using CycDesigN software. Three blocks are adopted with each parental/test genotype occurring in duplicate while the checks in triplicate for the entire experimental set up. Each plot constitutes three plants per genotype.
SHAPE \* MERGEFORMAT
Figure SEQ Figure \* ARABIC 1. P-rep lay out for Sigatoka disease screening for the available 51 parental lines including checks
Step 4: Setting up the experiment (Research technician, Research assistant and pathologist)
4.1. Screen house evaluations (artificial inoculation)
The Selected experimental site is prepared; which includes cleaning and disinfection if necessary.
Planting bags (Poly bags) containing sterilized mixture of loam soil, manure and sawdust mixed in a ratio of 3:1:1 respectively, are placed at the experimental site following the experiment setup.
Carefully transfer the weaned plants into planting bags (Polybags) following the experiment layout.
Plants are left to fully grow for 3 months with regular watering and monitoring and then inoculated with P. fijiensis.
4.2. Pathogen isolation and maintenance
4.2.1. Sample collection
Samples are taken from leaves of diseased banana plants from the field consisting of leaf sections bearing numerous black Sigatoka disease lesions at Stages 2-4 ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"ISBN":"9789788444800","author":[{"dropping-particle":"","family":"Lapeyre","given":"Luc","non-dropping-particle":"de","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Four","given":"Eric","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Guillermet","given":"Claire","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"FruiTrop Focus","id":"ITEM-1","issued":{"date-parts":[["2017"]]},"number-of-pages":"220-229","title":"Banana diseases and pests","type":"book"},"uris":["http://www.mendeley.com/documents/?uuid=2f015fb2-e049-40a5-ae9d-0ed8a71b102a"]}],"mendeley":{"formattedCitation":"(de Lapeyre et al., 2017)","manualFormatting":"(Lapeyre et al., 2017)","plainTextFormattedCitation":"(de Lapeyre et al., 2017)","previouslyFormattedCitation":"(de Lapeyre et al., 2017)"},"properties":{"noteIndex":0},"schema":"https://github.com/citation-style-language/schema/raw/master/csl-citation.json"}(Lapeyre et al., 2017).
4.2.2. Pathogen isolation
Several protocols / methods can be used to obtain pure isolates of Pseudocercospora species ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"ISBN":"9789788444800","author":[{"dropping-particle":"","family":"Lapeyre","given":"Luc","non-dropping-particle":"de","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Four","given":"Eric","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Guillermet","given":"Claire","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"FruiTrop Focus","id":"ITEM-1","issued":{"date-parts":[["2017"]]},"number-of-pages":"220-229","title":"Banana diseases and pests","type":"book"},"uris":["http://www.mendeley.com/documents/?uuid=2f015fb2-e049-40a5-ae9d-0ed8a71b102a"]}],"mendeley":{"formattedCitation":"(de Lapeyre et al., 2017)","manualFormatting":"(Lapeyre et al., 2017)","plainTextFormattedCitation":"(de Lapeyre et al., 2017)","previouslyFormattedCitation":"(de Lapeyre et al., 2017)"},"properties":{"noteIndex":0},"schema":"https://github.com/citation-style-language/schema/raw/master/csl-citation.json"}(Lapeyre et al., 2017) (Refer to annex 2)
4.2.3. Inoculum preparation
Inoculations can either be done using mycelial suspension or conidial suspension (Refer to annex 3)
4.2.4. Artificial inoculation of banana plants ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1051/fruits:2008030","ISSN":"02481294","abstract":"Introduction. This protocol aims at (i) analysing the nature of host-parasite interactions (resistance or susceptibility), and (ii) studying the variability of the pathogenicity of different pathogen populations (intra/inter-specific) with regard to both virulence and aggressiveness using artificial inoculation with Mycosphaerella fijiensis or Mycosphaerella musicola on plants and banana leaf pieces. The principle, key advantages, starting plant material and time required for the method are presented. Materials and methods. Laboratory materials required and details of the sixteen steps of the protocol (conidial inoculum preparation, artificial inoculation on banana plants and artificial inoculation on banana leaf pieces) are described. Possible troubleshooting is discussed. Results. Typical symptoms of the different diseases are obtained after artificial inoculation. 2008 CIRAD EDP Sciences.","author":[{"dropping-particle":"","family":"Abadie","given":"Catherine","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Zapater","given":"Marie Franoise","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Pignolet","given":"Luc","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Carlier","given":"Jean","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Mourichon","given":"Xavier","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Fruits","id":"ITEM-1","issue":"5","issued":{"date-parts":[["2008","9"]]},"page":"319-323","title":"Artificial inoculation on plants and banana leaf pieces with Mycosphaerella spp., responsible for Sigatoka leaf spot diseases","type":"article-journal","volume":"63"},"uris":["http://www.mendeley.com/documents/?uuid=5c6d11de-32c5-3ae8-b160-95bfa8b9cf0c"]}],"mendeley":{"formattedCitation":"(Abadie et al., 2008)","plainTextFormattedCitation":"(Abadie et al., 2008)","previouslyFormattedCitation":"(Abadie et al., 2008)"},"properties":{"noteIndex":0},"schema":"https://github.com/citation-style-language/schema/raw/master/csl-citation.json"}(Abadie et al., 2008)
Inoculate the youngest leaf on the lower surface using 1 ml of a spore suspension of 1 x 105 spores /ml concentration using painters brush / camel brush or microsprayer
Allow the inoculated area to dry for 1-2 hrs and then place plants into a humid chamber with 100% humidity for 48-96 hrs.
Move the plants into the screen house and monitor plants for infection by studying the inoculated leaves for symptoms on a 5-day interval.
4.2.5. Data collection
Daily monitoring should be done to capture the time of appearance of the first symptom (small light green dots or dashes of approximately 1 mm in length). Data collection starts when the first symptom appears and is done on a 5-day interval.
Data captured includes:
Incubation time (Time between inoculation with pathogen and appearance of symptoms)
Disease severity after Gauhl scoring scale (Table 1).
Symptom evolution time (Time from appearance of first symptom to development of mature spots).
Symptom stages ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"URL":"https://www.amazon.com/Bananas-Tropical-Agriculture-Robert-Stover/dp/0470206845","accessed":{"date-parts":[["2021","5","6"]]},"id":"ITEM-1","issued":{"date-parts":[["0"]]},"title":"Bananas (Tropical Agriculture Series): Stover, Robert H., Simmonds, N. W.: 9780470206843: Amazon.com: Books","type":"webpage"},"uris":["http://www.mendeley.com/documents/?uuid=d17b41dc-7a50-3c28-a09f-3bcaf93b210b"]}],"mendeley":{"formattedCitation":"(Bananas (Tropical Agriculture Series): Stover, Robert H., Simmonds, N. W.: 9780470206843: Amazon.Com: Books, n.d.)","manualFormatting":"(Stover and Simmonds, 1987)","plainTextFormattedCitation":"(Bananas (Tropical Agriculture Series): Stover, Robert H., Simmonds, N. W.: 9780470206843: Amazon.Com: Books, n.d.)","previouslyFormattedCitation":"(Bananas (Tropical Agriculture Series): Stover, Robert H., Simmonds, N. W.: 9780470206843: Amazon.Com: Books, n.d.)"},"properties":{"noteIndex":0},"schema":"https://github.com/citation-style-language/schema/raw/master/csl-citation.json"}(Stover and Simmonds, 1987)
Table 1. Disease Severity after Gauhl scoring scale ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"ISSN":"2224-4697","abstract":"Components of resistance were evaluated in seven Musa genotypes artificially inoculated with mycelia fragment suspensions of Mycosphaerella fijiensis Morelet (strain CCIBP-Pf80). Incubation period, number and area of necrotic lesions, infection index, area under the disease progress curve, asexual latent period, and number of spermogonia were evaluated in the genotype leaves to dissect the infective cycle of the fungus under greenhouse conditions. Incubation period in the inoculated leaves began at 14-21 days post infection, and significant differences were detected in the response of the different genotypes. Necrotic lesions were only observed in Grande naine, Pisang Awak, and significantly less in Pisang lilin. Grande naine reached the highest percentage of leaflet area with necrotic tissue, followed by Pisang Awak and Pisang lilin. Grande naine and Pisang Awak reached the greatest areas under the disease progress curves, while the lowest values were calculated in FHIA-18 and FHIA-25. The asexual latent period in Grande naine and Pisang Awak was significantly shorter (approximately 14 days) than in Pisang lilin. Greater numbers of spermogonia were observed in Grande naine and Pisang Awak, followed by Pisang lilin. Conidia were only detected in Grande naine, Pisang Awak and, in a significantly less number, in Pisang lilin. The different response observed of Musa spp. genotypes to the causal agent of Black Sigatoka indicated that the components of resistance used allowed the quantitative assessment of their reaction to this fungus. These results could improve or facilitate the efficiency and precision of the early evaluation process in banana and plantain breeding programs.","author":[{"dropping-particle":"","family":"Leiva-Mora","given":"Michel","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Alvarado-Cap","given":"Yelenys","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Acosta-Surez","given":"Mayra","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Cruz-Martn","given":"Mileidy","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Roque","given":"Berkis","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Mena Mndez","given":"Eilyn","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Rev. Proteccin Veg","id":"ITEM-1","issue":"1","issued":{"date-parts":[["2015"]]},"page":"60-69","title":"Components of resistance to assess Black Sigatoka response in artificially inoculated Musa genotypes","type":"article-journal","volume":"30"},"uris":["http://www.mendeley.com/documents/?uuid=4babfc70-4ee0-3089-8b97-dc4e3d1c7f67"]}],"mendeley":{"formattedCitation":"(Leiva-Mora et al., 2015)","plainTextFormattedCitation":"(Leiva-Mora et al., 2015)","previouslyFormattedCitation":"(Leiva-Mora et al., 2015)"},"properties":{"noteIndex":0},"schema":"https://github.com/citation-style-language/schema/raw/master/csl-citation.json"}(Leiva-Mora et al., 2015)
Rating Description 0
1
2
3
4
5
6No visible symptoms of the disease
Less than 1% (only streaks or up to ten spots of the leaf with disease symptoms)
1 to 5% of the leaf area with symptoms
6 to 15% of the leaf area with symptoms
16 to 33% of leaf area with symptoms
34 to 50% 0f the leaf area with symptoms
51 to 100% of the leaf area with symptoms
4.3. Field evaluations
The experimental fields are established in areas where the disease pressure is high for natural infection. NB spreader rows of highly susceptible cultivars should be planted around and within the experiment.
There should be a 2.5 m space between plants in each row and 3 m between rows.
The trials should be managed according to the local farmers agronomic practices. Leaf spot diseases should not be controlled.
The data should be collected for two successive cycles (on the mother plant and first sucker).
The evaluation of resistance begins six months after planting ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"author":[{"dropping-particle":"","family":"Kimunye","given":"Janet","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Jomanga","given":"Kennedy","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Tazuba","given":"Anthony Fredrick","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Were","given":"Evans","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Viljoen","given":"Altus","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Swennen","given":"Rony","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Mahuku","given":"George","non-dropping-particle":"","parse-names":false,"suffix":""}],"id":"ITEM-1","issued":{"date-parts":[["2021"]]},"title":"Genotype X Environment Response of Matooke Hybrids ( Naritas ) to Pseudocercospora fijiensis , the Cause of Black Sigatoka in Banana","type":"article-journal"},"uris":["http://www.mendeley.com/documents/?uuid=38b43202-434f-425a-866d-d866015a76fe"]}],"mendeley":{"formattedCitation":"(Kimunye et al., 2021)","manualFormatting":"(Kimunye et al., 2021)","plainTextFormattedCitation":"(Kimunye et al., 2021)","previouslyFormattedCitation":"(Kimunye et al., 2021)"},"properties":{"noteIndex":0},"schema":"https://github.com/citation-style-language/schema/raw/master/csl-citation.json"}(Kimunye et al., 2021)
Every test plant, except the extra plants of the guard and spreader rows, should be used for data collection.
4.3.1 Data Collection
Data collection should begin six months after planting.
Data is captured at different growth stages on each plant i.e., vegetative stage, flowering stage and at harvesting stage. three months until the plant is harvested.
Data is collected every after 3 months until the plant is harvested
Data at flowering and at harvest should also be captured for every plant
The following traits on each plant are captured: Total number of standing leaves (NSL), Total number of functional leaves (NFL), Youngest leaf with streaks (YLStr), Youngest leaf spotted (YLS), Disease severity index (DSI)
Immediately Save and Export the weekly data collected from the Field book App into a Gmail Account/Dropbox/OneDrive or any other storage option for back up purposes
Step 5. Data Curation and Analysis
5.1 Disease grade
Disease grades should be recorded for each leaf on each test plant. Only upright leaves should be recorded (with petioles upright). Leaves should be graded using Gauls modification of Stovers severity score system.
0 Healthy
1 < 1% leaf area affected (streaks and until 10 spots)
2 Until 5% of necrotic area
3 Between 6 and 15% of necrotic area
4 Between 16 and 33% of necrotic area
5 Between 34 and 50% of necrotic area
6 > than 51% of necrotic area
5.2 Disease severity index (DSI)
Disease severity is the amount of leaf area affected by Pseudocercospora leaf spots and can be expressed in disease grades or in percentage.
The black Sigatoka disease severity index for each plant is computed u s i n g t h e f o l l o w i n g f o r m u l a : D S I = " [ n b ( N " 1 ) T ] 1 0 0
W h e r e :
n = n u m b e r o f l e a v e s i n e a c h d i s e a s e s e v e r i t y g r o u p / g r a d e ,
b = s e v e r i t y g r o u p / g r a d e ;
N = n u m b e r o f s e v e r i t y g r o u p s u s e d i n t h e s c a l e ( 7 ) ;
T = t o t a l n u m b e r o f l e a v e s s c o r e d .
5 . 3 I n d e x o f n o n - s p o t t e d l e a v e s
T h e p r o p o r t i o n o f h e a l t h y l e a v e s o r I n d e x o f n o n - s p o t t e d l e a v e s ( I N S L ) i s e s t i m a t e d a s t h e p r o p o r t i o n o f l e a v e s r a t e d i n g r a d e 0 o f t h e t o t a l l e a v e s a s s e s s e d .
T h e i n d e x o f n o n - s p o t t e d l e a v e s i s c o m p u t e d f o r e a c h p l a n t u s i n g t h e f o l l o w i n g f o r m u l a e : I N S L = [ ( Y L S " 1 ) N S L ] 1 0 0
W h e r e :
Y L S = r a n k o f t h e y o u n g e s t l e a f c o u n t i n g f r o m b o t t o m b e a r i n g a t l e a s t 1 0 n e c r o t i c l e s i o n s w i t h a d r y c e n t r e ,
N S L = t h e n u m b e r o f l e a v e s s t a r t i n g f r o m t h e y o u n g e s t ,
I N S L = t h e p r o p o r t i o n o f s t a n d i n g leaves without the typical black Sigatoka late-stage necrotic lesions.
5.4 Area under disease progress curve (AUDPC)
The disease severity index (DSI) at different evaluation times is used to calculate the area under the disease progress curve (AUDPC) per cycle, using the formula:
A U D P C = "n i 1 [ ( X i + 1 + X i ) 2 ] [ t i + 1 " t i ]
W h e r e X i = d i s e a s e s e v e r i t y i n d e x a t i t h d a y ,
t i = t h e t i m e i n d a y s a f t e r a p p e a r a n c e o f t h e d i s e a s e a t i t h d a y ,
n = t h e t o t a l n u m b e r o f o b s e r v a t i o n s .
7 . R e f e r e n c e s
A D D I N M e n d e l e y B i b l i o g r a p h y C S L _ B I B L I O G RAPHY Abadie, C., Zapater, M. F., Pignolet, L., Carlier, J., & Mourichon, X. (2008). Artificial inoculation on plants and banana leaf pieces with Mycosphaerella spp., responsible for Sigatoka leaf spot diseases. Fruits, 63(5), 319323.
Bananas (Tropical Agriculture Series): Stover, Robert H., Simmonds, N. W.: 9780470206843: Amazon.com: Books. (n.d.). Retrieved May 6, 2021, from https://www.amazon.com/Bananas-Tropical-Agriculture-Robert-Stover/dp/0470206845
Carlier, J., Zapater, M. F., Lapeyre, F., Jones, D. R., & Mourichon, X. (2000). Septoria leaf spot of banana: A newly discovered disease caused (anamorph Septoria eumusae) by Mycosphaerella eumusae. Phytopathology, 90(8), 884890.
Chang, T. C., Salvucci, A., Crous, P. W., & Stergiopoulos, I. (2016). Comparative Genomics of the Sigatoka Disease Complex on Banana Suggests a Link between Parallel Evolutionary Changes in Pseudocercospora fijiensis and Pseudocercospora eumusae and Increased Virulence on the Banana Host. PLoS Genetics, 12(8).
de Lapeyre, L., Four, E., & Guillermet, C. (2017). Banana diseases and pests. In FruiTrop Focus.
Irish, B. M., Goenaga, R., & Ploetz, R. C. (2006). Mycosphaerella fijiensis, Causal Agent of Black Sigatoka of Musa spp. Found in Puerto Rico and Identified by Polymerase Chain Reaction. Plant Disease, 90(5), 684.
Kimunye, J., Jomanga, K., Tazuba, A. F., Were, E., Viljoen, A., Swennen, R., & Mahuku, G. (2021). Genotype X Environment Response of Matooke Hybrids ( Naritas ) to Pseudocercospora fijiensis , the Cause of Black Sigatoka in Banana.
Leiva-Mora, M., Alvarado-Cap, Y., Acosta-Surez, M., Cruz-Martn, M., Roque, B., & Mena Mndez, E. (2015). Components of resistance to assess Black Sigatoka response in artificially inoculated Musa genotypes. Rev. Proteccin Veg, 30(1), 6069.
8. Annex: Forms/Templates to be used for monitoring and data collection
Annex 1: Flow Chart for Screening banana genotypes against P. Fijiensis (Black Sigatoka)
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Select leaf material containing Stage 2 lesions of suspected, or Stage 2-3 for P. fijiensis and cut them into 1-2 cm squares.
Immerse the tissue into a beaker containing 1% sodium hypochlorite for 1 min, remove and wash five times with sterile distilled water, blot dry using sterile blotting paper.
Using a sterile scalpel blade, make incisions on either side of the lesion, taking care not to cut right through the leaf piece.
Excise epidermal pieces of tissue (approximately 2 mm2), and plate onto PDA.
Seal the Petri dish with Parafilm and incubate at 25C for 10 days.
Transfer small portions (hyphal tips) of the culture to V8 media to induce conidia production.
Mount the conidia on a glass slide and observe under a compound microscope to identify Pseudocercospora species
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3.1 Development of mycelial suspension
From an actively growing monosporic culture on PDA, scrap mycelia directly into a sterile mortar with a surgical blade.
Grind in 5 ml sterile distilled water with a drop of 0.05% Tween 20 into a fine suspension. Filter the suspension through two layers of cheesecloth to get rid of media debris.
Quantify the suspension using a haemocytometer.
Alternatively, harvested mycelia can be weighed on a balance, and fragmented by blending in sterile distilled water.
3.2 Development of spore suspension
Add 3 ml of sterile distilled water with a drop of 0.05% Tween 20 to a sporulating culture (sporulating colonies appear pinkish).
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