Improvement of Banana for Smallholder Farmers in the
Improvement of Banana for Smallholder Farmers in the Great Lakes Region of Africa WP 2: Pest and diseases report 30 September 2016 - 01 April 2017
Diseases and pests in east African bananas Fusarium wilt Sigatoka diseases Nematodes Banana weevil
Primary outcomes Accelerated Matoke and Mchare banana breeding through early identification of material resistant to Fusarium wilt, Sigatoka, nematodes and weevils • Determine the relative importance of Fusarium oxysporum f. sp cubense, Mycosphaerella spp. , nematodes and weevil populations as targets for selection breeding in East Africa • Screen selected EAHB, and Mchare diploids and NARITA hybrids for resistance to Fusarium wilt, Sigatoka, nematodes and weevils and determine influence of plant development, environmental conditions and season on disease severity • Rapid and precise screening methods for Fusarium wilt, Sigatoka, nematodes and weevils developed for use in breeding programs • Train staff in banana disease evaluation, resistance screening and pathogen and pest identification with focus on Fusarium wilt, Sigatoka, nematodes and weevils
Intermediate Outcomes Determine the relative importance of Fusarium oxysporum f. sp. cubense, Mycosphaerella spp. , nematodes and weevil populations as targets for selection breeding in ECA Outputs Pests and diseases characterised in breeding and testing sites and screening banana cultivars for resistance to Fusarium wilt, Sigatoka, nematodes and weevils Targets/ Milestones YEAR 1 YEAR 2 Assess impact Determine (disease score) distribution and for at least 20 impact of target plants for each pests and site of target diseases across pest and sites in Tanzania disease species and Uganda on different using banana morphological varieties, and molecular across different diagnostics and environments pathogenicity/ and collect virulence tests representative samples Appoint and train Ph. D student for Fusarium wilt and Mycosphaerella spp. Progress Variance YEAR 3 Catalogue distribution and impact of pest and pathogens for different banana varieties across different environments of Tanzania and Uganda Characterise species and populations of target pests and diseases recovered from visits to screening trials on station and at regional testing sites using morphological and molecular diagnostics and pathogenicity/ virulence tests • Sampling completed at the five screening sites. • Characterization of 208 isolates has been completed up to VCG level. • Different varieties affected by Foc has been catalogued. • • 5% variance Reason: 26 Foc isolates still to be characterised. Results will be available in the next reporting period. Sampling from five screening sites is complete. 50% variance. PCR detection of P. fijiensis from 920 infected leaves Reasons: Identification of other species delayed by a lack of • Failure by published positive controls to verify specificity and utility of PCR primers to amplify primers. Type strain isolates have been received from • Slow growth of fungus CBS and the activity is in progress. delays isolation and Characterisation of isolates recovered from Kawanda characterisation and Sendusu done up to mating type process Isolation from regional testing trials on going Optimisation of SSR markers for further characterisation is in progress Virulence testing is pending genetic characterisation • Nematode samples from soils and banana roots were collected in three sites; Mbeya, Kagera and Arusha for mapping nematodes species abundance and distribution in Tanzania. Mapping of their distribution is in progress. 30% variance. Reason: Survey done in Tanzania only. NARO will collect nematodes in Uganda • In Uganda 13 sites were visited, and survey data collected from five banana farms per site. Data include GPS readings, cultivars on the farm, corm damage assessment and banana weevil trap catches. Results show that the intensity of the banana weevil damage decreased with elevation. • A survey team has been constituted and trained to collect samples from Tanzania. 20% variance. Reason: Transfer of funds to facilitate the team in Tanzania has failed twice.
VCG groups or VCG complexes of Foc identified in the five screening sites and varieties affected. Site Variety VCG group/Complex 0124 Kawanda 0128 01212 01220 1 1 3 2 Sukari Ndizi 1 1 Pisang Kepok Percentage 5 11 1 10 15 15 5 25 2 7 11 2 9 11 2 1 Pisang Awak Total 1 4 2 MV 1 F 1 All cultivars 1 1 Pisang Awak Kagera Total 1 Mshares Sukari Ndizi 0124/5/8/22 1 Sukari Ndizi Mbeya 0124/22 5 Safeti Velchi Embu Arusha 01222 Pisang Awak Sukari Ndizi Mbarara 0125 44 8 4 1 1 5 53 1 3 5 17 11 5 4 6 1 9 13 6 55 19 5 5 63 1 16 58 41 206 9. 2 2. 4 30. 6 0. 5 7. 8 28. 2 18. 9 100. 0
Intermediate Outcomes Determine the relative importance of Fusarium oxysporum f. sp. cubense, Mycosphaerella spp. , nematodes and weevil populations as targets for selection breeding in ECA Outputs Collect and preserve Fusarium wilt, Sigatoka, nematodes and weevils from each breeding and testing site for characterization and rapid screening of bananas Stellenbosch univ (Fusarium), Sigatoka (IITA), weevil (NARO), nematodes (ARITengeru) Targets/ Milestones YEAR 1 YEAR 2 YEAR 3 Recover from each breeding and testing sites sample pest and disease isolates and culture into pure stocks for each accession For each accession preserve using appropriate methods for long term storage Establish reference collections at NARO, IITA and SUN and wherever possible replicate accessions across these collections Recover from each sample collected from screening trials on station and from regional testing trials pest and disease isolates, culture into pure stocks and preserve each accession Progress Variance • 208 Foc samples collected in Mbeya, Arusha, Mbarara and Kawanda were added to the culture collection. 0% variance • 40 pure isolates of P. fijiensis have been made and put into long term storage at Kawanda, Uganda. • Isolation from Mbeya on going while from other sites, fresh samples will be collected in May-June. 40% variance Reason: Samples collected originally failed to discharge spores. Lessons learned – isolations have to be done from freshly collected samples • Culture of nematode species of 30% variance: Radopholus similis and Pratylenchus Reason: Survey has been goodeyi in vitro in pots and carrot-disc conducted only in cultures are maintained at HORTI Tanzania. Currently Tengeru and SRI Kibaha for field NARO is collecting banana screening activities. nematode samples in Uganda. Nematodes will be extracted, identified and culture will be established and maintained both in pot and in carrot-discs. • Banana weevils were captured and 0% variance reared on detached banana corms. Eight banana weevil populations are currently being maintained at Kawanda
Intermediate Outputs Outcomes Determine the Rapid and accurate relative importance methods to of Fusarium determine the oxysporum f. sp. identity and fitness cubense, of Fusarium wilt, Mycosphaerella Sigatoka, spp. , nematodes and weevil populations weevils developed as targets for and validated selection breeding in ECA Targets/ Milestones YEAR 1 YEAR 2 Fully document (characterise and preserve) at least 25 accessions from each target pest and pathogen acquired from surveys of Tanzania and Uganda Fully document (characterise and preserve) at least 6 accessions from each target pest and pathogen acquired from on station trials and from regional test sites Progress YEAR 3 Fully document (characterise and preserve) at least 6 accessions from each target pest and pathogen acquired from on station trials and from regional test sites Variance All isolates collected were fully documented. 0% variance Markers were developed for the rapid identification of Foc Lineage VI strains. The markers were used to identify samples collected at the screening sites. A paper on markers was prepared for publication. • Preserved P. fijiensis isolates from Kawanda typed using mating type primers. • Further characterisation to start in May 50% variance Reason: Isolation from other sites failed due to loss of spores on storage. The activity to obtain fresh samples for isolation is on going • The experiment has been established in 50% variance: the screen house at the HORTI-Tengeru Reason: Data Arusha to screen 20 NARITA banana from collection has not yet mother trial site and two local checks (Ndizi commenced. Ngombe and Williams) for nematode resistance using multiple species screening method. • All isolates collected were documented pending is the molecular characterisation, 20% variance Reason: primers that were ordered have not been delivered
Detection of Pseudocercospora fijiensis using DNA extracted directly from infected leaves and using ITS species specific primers MF 137/R 635.
Intermediate Outputs Outcomes Determine the Map the distribution of relative importance Fusarium wilt, of Fusarium Sigatoka, nematodes oxysporum f. sp. and weevils of banana cubense, and using GIS link to Mycosphaerella spp. , climatic and nematodes and environmental data weevil populations as targets for selection breeding in ECA Targets/ Milestones YEAR 1 Survey and secondary data used to develop models to account for economic value of pests and diseases in Matoke and Mchare YEAR 2 Selection weights assigned to pests and diseases based on economic value of pests and diseases in bananas YEAR 3 Analyse data catalogued from surveys of Tanzania and Uganda on distribution and impact of pest and pathogens for different banana varieties across different environments Selection Index for pest/disease resistance progressively incorporated into selection of Matoke and Mchare hybrids at PYT Progress Variance • All data on the distribution and impact 10% variance of Fusarium wilt in Uganda and Reason: Mapping will Tanzania has been collected. be completed when all data is available. • Data from the surveys is analysed 30% variance and maps are being generated. Reason: Identification • Information generated is accurate for of Pseudocercospora P. fijiensis, the species that has species causing leaf positively been confirmed using spots was delayed molecular markers. due to primers failing. • Work in progress to type samples for There are plans to the other species associated with develop another set Sigatoka leaf spots. of primers. • Only survey for nematodes distribution has been effected in Tanzania and mapping of nematode distribution in Tanzania from GPS coordinated is in progress 50% variance Reason: Mapping will be completed once data from all diseases is available. • Data have been catalogue 10% variance Reason: Mapping has not been completed because data from all diseases need to be combined and the relevance of each disease calculated.
Intermediate Outcomes Screen selected EAHB hybrids and Mchare diploids and NARITA hybrids for resistance to Fusarium wilt, Sigatoka, nematodes and weevils and determine influence of plant development, environmental conditions and season on disease severity Outputs Targets/ Milestones YEAR 1 YEAR 2 YEAR 3 Map natural populations Map distribution and • and impact (disease of Fusarium wilt, (disease score for at least 20 Sigatoka, nematodes at least 20 plants per site and weevils on station per site and banana variety) of • and at regional field banana variety) of target pest and pathogen testing sites in Uganda target pest and pathogen populations and Tanzania and populations collected from visits • determine influence of collected from on station and to plant development, surveys visits on station regional test sites environmental and to regional test sites conditions and season on disease severity Progress Variance Two samples from NARITA plants at 0% variance Arusha have been received and were identified Identification of Foc isolates from trial sites will continue for the next 2 years Evaluations of Sigatoka at regional testing sites has been completed for Mbeya, Arusha and Kawanda. Samples of diseased leaves have been collected from evaluated plants and are being tested to confirm pathogen identity. • Mapping of nematode distribution in Tanzania is progressing while nematode sampling in Uganda is in progress • No data has been received on banana weevil from regional testing sites. 50% variance Reason: Regional trials were planted late and 2016/2017 was extremely dry. This affected plant establishment and symptom expression: 50% variance Reason: Mapping in Tanzania in progress. Sampling of nematodes in Uganda in progress 50% variance Reason: Data on weevils from regional testing sites not yet available since the plants have not yet been harvested
Leaf spot severity in low altitude (<1200 m a. s. l), mid altitude (1201 -1500) and high altitude (>1500 m a. s. l) ranges in Uganda and Tanzania 50 Disease severity index(%) 45 40 35 30 25 Uganda 20 15 10 Tanzania 5 0 Low Altitude Mid Altitude ranges High Altitude
Intermediate Outcomes Screen selected EAHB hybrids and Mchare diploids and NARITA hybrids for resistance to Fusarium wilt, Sigatoka, nematodes and weevils and determine influence of plant development, environmental conditions and season on disease severity Outputs YEAR 1 Trials being Evaluate resistance of prepared EAHB hybrids on station and determine influence of plant development, environmental conditions and season on disease severity caused by Fusarium wilt, Sigatoka, nematodes and weevils Targets/ Milestones YEAR 2 YEAR 3 Progress Complete biannual visits on station to assess impact of target pest and disease species on • EAHB hybrids Collect samples of target pests and diseases for preservation in reference collections and to characterise populations Not relevant to Fusarium wilt, as it does not cause disease to EAHB. Variance 0% variance Evaluation of EAHB hybrids EAHB 0% variance hybrids evaluated in Sendusu on a quarterly basis and samples collected, isolated and preserved • The experiment has been established in the screen house at the HORTI-Tengeru Arusha to screen Ndizi ng’ombe, Mchare and 20 NARITA banana from mother trial site for nematode resistance using multiple species screening method. 30% variance Reason: The same experiment will be established in the field • Breeding stations in Uganda (Kawanda and Sendusu) were surveyed to determine the impact of the banana weevil on banana breeding materials. Mchare bananas appear to be least infested with the banana weevil. 0% variance
N A N RIT A A N RIT 01 A A N RIT 02 A A N RIT 03 A A N RIT 04 A A N RIT 05 A A N RIT 06 A A N RIT 07 A A N RIT 08 A A N RIT 09 A A N RIT 10 A A N RIT 11 A A N RIT 12 A A N RIT 13 A A N RIT 14 A A N RIT 15 A A N RIT 16 A A N RIT 17 A A N RIT 18 A A N RIT 19 A A N RIT 20 A A N RIT 21 A A N RIT 22 A A N RIT 23 A A N RIT 24 AR A IT 25 A 26 Mean DSI (%) Sigatoka severity index computed based on severity per leaf as described 50 45 40 35 30 25 20 15 10 5 0 Cultivar
Motooke cultivar e w ez i am ba ra ra w R am N be an d am N ka yi te m ak i N ga on in ak N e er aw ira lu lu ab ay ak N N ak as ak N e m om a ak ab u N e ns a iru lo g po M az M bw Ki sa kw e gy ng u ra ra zi Ka cu bu a ra hi m te ye ak N Ka A IIT ba ku tu En ra zi En % total cross section damage Damage due to banana weevils (Natural infestation) on Matooke genotypes at Kawanda 80 70 60 50 40 30 20 10 0
Intermediate Outcomes Screen selected EAHB hybrids and Mchare diploids and NARITA hybrids for resistance to Fusarium wilt, Sigatoka, nematodes and weevils and determine influence of plant development, environmental conditions and season on disease severity Outputs YEAR 1 Trials being Evaluate resistance of prepared selected Mchare diploids on station and determine influence of plant development, environmental conditions and season on disease severity caused by Fusarium wilt, Sigatoka, nematodes and weevils Targets/ Milestones YEAR 2 Progress Variance YEAR 3 Complete biannual visits on station to assess impact of target pest and disease species on Mchare diploids Collect samples of target pests and diseases for preservation in reference collections and to characterise populations • Screening and field trials are 90% variance being established at Kawanda in Reason: A delay in Uganda and Arusha in Tanzania. the multiplication and planting of Mshare diploids. Trials to be established in March and April 2017. • 8 Mchares have been evaluated 0% variance at least 3 times in Kawanda and samples collected. • Plantlets are in the screen house at their final growth stage before taken to the field for experiment trial establishment 60% variance Reason: The experiment is not yet planted • Screening and field trials are 90% variance being established at Kawanda in Reason: A delay in Uganda and Arusha in Tanzania. the multiplication and planting of Mshare diploids.
Intermediate Outputs Outcomes YEAR 1 Trials being Screen selected Evaluate resistance of prepared EAHB hybrids and 27 NARITA hybrids Mchare diploids and under varied field NARITA hybrids for conditions at 3 regional resistance to field test sites and Fusarium wilt, determine influence of Sigatoka, nematodes plant development, and weevils and environmental determine influence conditions and season of plant development, on disease severity environmental caused by Fusarium conditions and wilt, Sigatoka, season on disease nematodes and weevils severity Targets/ Milestones YEAR 2 Variance • Data collected from NARITA hybrids started in October 2016. • At an early stage hybrids have not yet been affected by Fusarium wilt 10% variance Reason: First data was made available only in March 2017. YEAR 3 Complete biannual visits to regional test sites to assess impact of target pest and disease species on 27 NARITA hybrids Collect samples of target pests and diseases for preservation in reference collections and to characterise populations Progress Collect samples of target pests and diseases for preservation in reference collections and to characterise populations • Data collection from NARITA hybrids started in October 2016. 30% variance Reason: Trials were established late and the season was dry. This affected plant establishment and disease progression. • The experiment has been 40% variance established in the screen house Reason: Data at the HORTI-Tengeru Arusha to collection has not yet screen 20 NARITA banana from commenced mother trial site and two local checks (Ndizi ng’ombe and Mchare) for nematode resistance using multiple species screening method. • The data on banana weevil infestation at regional testing not yet available 10% variance Reason: Weevil infestation assessment expected after the NARITAS have been harvested
Intermediate Outcomes Screen selected EAHB hybrids and Mchare diploids and NARITA hybrids for resistance to Fusarium wilt, Sigatoka, nematodes and weevils and determine influence of plant development, environmental conditions and season on disease severity Outputs In vitro screening methods for Fusarium wilt, Sigatoka, nematodes and weevils developed to increase the speed and number of samples that can be evaluated in breeding programs Targets/ Milestones YEAR 1 YEAR 2 YEAR 3 MSc student Test suitability of Challenge selected recruited to in vitro methods banana varieties characterize and across target with representative partition pests and populations of resistance to pathogens for at target pests and nematodes and least 10 pathogens (from weevils in Musa selected banana surveys) and Existing biossays varieties document results for Foc, Mycosphaerella Bioassays for Factors spp. , nematodes Foc, determining and weevils Mycosphaerella resistance to target catalogued spp. , pests and diseases nematodes and in selected Develop test runs weevils adapted/ cultivars to assess survival developed for determined (such of banana high throughput as stomatal material in vitro screening. characteristics and using different epicutular wax for methods and to Mycosphaerella ensure suitability spp. ) across varieties Progress Variance A range of techniques for a small plant screening test have been investigated. Inoculum levels and inoculation methods have been optimised. Six known phenolic compounds were identified in banana, as well as four unknown constitutive and three unknown induced phenolic acids. All known phenolic compounds suppressed Foc at a concentration of 2. 5 m. M 70% variance Reason: The 19 cultivars from ITC arrived in March 2017. They will now be multiplied, and it is expected that the trial will be complete by June 2018. • Field trial with selected varieties was 50% variance established in Sendusu in December 2016 to Reason: There determine components of resistance. Screen was a delay in house experiments established with suckers getting tissue to optimise inoculum type and level will be culture plants inoculated in May. • Multiple species screening method for 0% variance nematode resistance has been adopted and is in use in the screen house experiment • A field trial of 10 diploid bananas, a control 0% variance and 2 triploid bananas has been established to characterize and partition resistance to banana weevils in Musa • The plants have been challenged (infested with 10 weevils per mat)
Mean percentage suppression of Fusarium oxysporum f. sp. cubense by plant phenolic compounds.
Intermediate Outcomes Rapid and precise screening methods for Fusarium wilt, Sigatoka, nematodes and weevils developed for use in breeding programs Outputs Compare the reliability of young plant resistance testing compared to whole plant testing in field for evaluations against Fusarium wilt, Sigatoka, nematodes and weevils Targets/ Milestones YEAR 1 YEAR 2 YEAR 3 Establish cultivation of plantlets of selected banana varieties Manage cultivation of plants of selected banana varieties Challenge whole plants of selected banana varieties with representative populations of target pests and pathogens from surveys Progress Variance • Mshare bananas will be evaluated in greenhouse and field. Results will be correlated to assess the reliability of young plant screening compared to field evaluations. 80% variance Reason: Trials will be planted in April and May 2017 • Eight differential cultivars selected for 80% variance virulence testing are under tissue culture Reason: Delay in multiplication getting tissue culture plants. • Screen house experiment for nematodes 60% variance screening is progressing Reason: Field experiment need to be established • Established a field screening trial for 80% variance resistance to banana weevils using three Reason: Trials need resistant cultivars; three susceptible to be planted in April cultivars; and four NARITA hybrids and May 2017 whose response to banana weevils is unknown. • Results from the corm damage due to weevils for the first and second ratoon cycles will be compared with the results of the improved banana larvae development on detached corm bioassay to determine the reliability of the short screening method and test for suitability for high throughput.
Intermediate Outcomes Train staff in banana disease evaluation, resistance screening and pathogen and pest identification with focus on Fusarium wilt, Sigatoka, nematodes and weevils Outputs Train staff on survey and culturing of Fusarium wilt, Sigatoka, nematodes and weevils Stellenbosch univ (Fusarium), Sigatoka (IITA), weevil (NARO), nematodes (ARITengeru). YEAR 1 Targets/ Milestones YEAR 2 Some institutions are lacking trained personnel to carryout survey for mapping the occurrence, distribution and abundance of identified key pests and pathogens Field manuals developed to support the recognition of target pests and diseases and protocols for sample collection Progress YEAR 3 On station training provided in both Tanzania and Uganda during visits A manual has been developed for identification of Foc and other pest and diseases. This has now been published. • A manual has been developed for identification of Sigatoka and other pest and diseases. This is being prepared for publication. • A manual has been developed for nematode sampling and other pest and diseases. This is being prepared for publication. • A manual has been developed for identification of banana and other pest and diseases. This is being prepared for publication. 4 technicians collecting data from breeding fields have been trained in Uganda. Variance 0% variance
Training enumerators on collecting sigatoka data and harmonize disease screening protocols.
Challenges encountered Financially: • The main challenge for the execution of the weevil components of WP activities has been the failure to transfer funds for the team in Tanzania Technically: • Multiplication of TC plants was delayed at Kawanda and Arusha due to holidays. • The delivery of banana plants from ITC was delayed by 6 mo. • Experiments on rapid screening protocols for Sigatoka was delayed due to losses of all TC plants a month before planting. So suckers were used to establish the trial. • Problems with primers for P. eumusae and P. musae delayed activities on identification. Cultures of Pseudocercospora were ordered from the CBS fungal collection. • Establishing pure cultures of Pseudocercospora spp. has been a major challenge, as the fungus grows very slowly and is outcompeted by saprophytes. The fungus also did not store very well on banana tissues and isolations need to be conducted soonest. • Transport of nematode samples across borders delayed collections in Uganda. • Routine management practices on older fields affected data collection for weevils. Communication: • A lack of communication from laboratory staff about progress on multiplication has caused delays in the establishment of Mshare trials.
Lessons learned • Report regular progress on the multiplication and plants availability for better planning of activities. • Given the prolonged delay in obtaining tissue culture plants and the slow growth of fungus in culture, proper planning is required to ensure timely delivery of expected outputs. It is good to have a backup plan, should things not go as planned. • Regular Skype meetings have been found very useful in tracing the development of activities between partners. • Preliminary findings indicate that 'Mshale' diploids are showing less damage due to the banana weevil. This implies that Mshale diploids can be a good source of resistance to banana weevil. • Detached banana corms on water-agar medium supplemented with 8 mg/L of GA 3 gained weight and retained colour after 30 days. This excised corm preservation will greatly improve the banana weevil bioassays since plant defence components will no longer be broken down during the assay. • Banana weevil damage was observed at high altitudes not previously observed like 1900 m above sea level in Uganda. This implies that the pest range for banana the weevils is increasing and evaluation of weevil resistant genotypes should consider such high altitude areas.
Other relevant project information • Travelling will become more extensive and expensive than originally anticipated to visit trial sites and other related activities such plant multiplication, field planting and data collection, • EAHB hybrids appear not to be affected by Fusarium wilt. Trials will thus be limited to the screening of NARITA hybrids and Mshare diploids.
Work plan Fusarium wilt: • Finish the identification and characterisation of Foc at the screening sites by end of 2017. • Establish Mshare trials in Uganda and Arusha in April and May respectively. • Multiply bananas in 2017 to be used for rapid screening technique development. Sigatoka • Finish the identification and characterisation of Pseudocercospora species and genetically characterise the isolates • Determine pathogenic variability among genetically different isolates and implication in breeding • Optimize inoculation protocol and develop a rapid screening protocol Nematodes: • Continue with in vitro nematodes culturing in pots and in carrot discs for timely use in banana screening experiments • HORTI-Tengeru to do weevil collections for Uganda in Tanzania as soon as they received sampling protocol • To map the GPS coordinates of the collection sites and indicate the significance of the nematodes in their collection sites. • Finish the nematode sampling and identification in Uganda for their occurrence and their distribution by end of 2017.
Summary 1. Significant progress has been made to get the project back on schedule. This include collections of pests and pathogens at all locations 2. Problems with Sigatoka isolations and identifications had been resolved. 3. Problems encountered with quarantine regulations for movement of weevils and nematodes across country borders were resolved. 4. Molecular and biochemical responses of banana varieties are being investigated in addition to small plant screening in order to rapid screening methods to support banana breeding. 5. Major delays were due to tissue culture problems and the availability of planting materials. This has now been largely resolved. 6. Communication within WP 2 has been significantly improved, but intergroup collaboration still needs improvement. 7. Several training sessions were presented and attended in the past year, with good outcomes to WP 2 and the entire breeding project.
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