1. WP2: Pest and diseases report 30 September 2016 - 01 April 2017
Improvement of Banana for Smallholder Farmers in the Great Lakes Region of Africa
WP2: Pest and diseases report
30 September April 2017

2. Diseases and pests in east African bananas
Fusarium wilt
Sigatoka diseases
Nematodes
Banana weevil

3. 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

4. Sampling completed at the five screening sites.
Intermediate Outcomes
Outputs
Targets/ Milestones
Progress
Variance
YEAR 1
YEAR 2
YEAR 3
Determine the relative importance of Fusarium oxysporum f.sp. cubense, Mycosphaerella spp., nematodes and weevil populations as targets for selection breeding in ECA
Pests and diseases characterised in breeding and testing sites and screening banana cultivars for resistance to Fusarium wilt, Sigatoka, nematodes and weevils
Assess impact (disease score) for at least 20 plants for each site of target pest and disease species on different banana varieties, across different environments and collect representative samples
Appoint and train PhD student for Fusarium wilt and Mycosphaerella spp.
Determine distribution and impact of target pests and diseases across sites in Tanzania and Uganda
using morphological and molecular diagnostics and pathogenicity/ virulence tests
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.
PCR detection of P. fijiensis from 920 infected leaves
Identification of other species delayed by a lack of positive controls to verify specificity and utility of PCR primers. Type strain isolates have been received from CBS and the activity is in progress.
Characterisation of isolates recovered from Kawanda and Sendusu done up to mating type
Isolation from regional testing trials on going
Optimisation of SSR markers for further characterisation is in progress
Virulence testing is pending genetic characterisation
50% variance.
Reasons:
Failure by published primers to amplify
Slow growth of fungus delays isolation and characterisation process
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.

5. VCG groups or VCG complexes of Foc identified in the five screening sites and varieties affected.
Variety
VCG group/Complex
0124
0125
0128
01212
01220
01222
0124/22
0124/5/8/22
Total
Kawanda
Pisang Awak 1
Sukari Ndizi 5 11
Mbarara
Safeti Velchi
Embu 4 10 15
Arusha
Mshares 3 2 25 7 9
Pisang Kepok
MV1F1
Mbeya 44 53 8 17
Kagera
All cultivars 6 13 55 19 63 16 58 41
206
Percentage
9.2
2.4
30.6
0.5
7.8
28.2
18.9
100.0

6. Intermediate Outcomes
Outputs
Targets/ Milestones
Progress
Variance
YEAR 1
YEAR 2
YEAR 3
Determine the relative importance of Fusarium oxysporum f.sp. cubense, Mycosphaerella spp., nematodes and weevil populations as targets for selection breeding in ECA
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 (ARI-Tengeru)
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
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 Radopholus similis and Pratylenchus goodeyi in vitro in pots and carrot-disc cultures are maintained at HORTI Tengeru and SRI Kibaha for field banana screening activities.
30% variance:
Reason: Survey has been conducted only in Tanzania. Currently NARO is collecting 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 reared on detached banana corms. Eight banana weevil populations are currently being maintained at Kawanda

7. All isolates collected were fully documented.
Intermediate Outcomes
Outputs
Targets/ Milestones
Progress
Variance
YEAR 1
YEAR 2
YEAR 3
Determine the relative importance of Fusarium oxysporum f.sp. cubense, Mycosphaerella spp., nematodes and weevil populations as targets for selection breeding in ECA
Rapid and accurate methods to determine the identity and fitness of Fusarium wilt, Sigatoka, nematodes and weevils developed and validated
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
All isolates collected were fully documented.
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.
0% variance
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 the screen house at the HORTI-Tengeru Arusha to screen 20 NARITA banana from mother trial site and two local checks (Ndizi Ngombe and Williams) for nematode resistance using multiple species screening method.
50% variance:
Reason: Data collection has not yet commenced.
All isolates collected were documented pending is the molecular characterisation,
20% variance
Reason: primers that were ordered have not been delivered

8. Detection of Pseudocercospora fijiensis using DNA extracted directly from infected leaves and using ITS species specific primers MF137/R635.

9. Intermediate Outcomes
Outputs
Targets/ Milestones
Progress
Variance
YEAR 1
YEAR 2
YEAR 3
Determine the relative importance of Fusarium oxysporum f.sp. cubense, Mycosphaerella spp., nematodes and weevil populations as targets for selection breeding in ECA
Map the distribution of Fusarium wilt, Sigatoka, nematodes and weevils of banana and using GIS link to climatic and environmental data
Survey and secondary data used to develop models to account for economic value of pests and diseases in Matoke and Mchare
Selection weights assigned to pests and diseases based on economic value of pests and diseases in bananas
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
All data on the distribution and impact of Fusarium wilt in Uganda and Tanzania has been collected.
10% variance
Reason: Mapping will be completed when all data is available.
Data from the surveys is analysed and maps are being generated.
Information generated is accurate for P. fijiensis, the species that has positively been confirmed using molecular markers.
Work in progress to type samples for the other species associated with Sigatoka leaf spots.
30% variance
Reason: Identification of Pseudocercospora species causing leaf spots was delayed due to primers failing. There are plans to develop another set 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
Reason: Mapping has not been completed because data from all diseases need to be combined and the relevance of each disease calculated.

10. Intermediate Outcomes
Outputs
Targets/ Milestones
Progress
Variance
YEAR 1
YEAR 2
YEAR 3
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
Map natural populations of Fusarium wilt, Sigatoka, nematodes and weevils on station and at regional field testing sites in Uganda and Tanzania and determine influence of plant development, environmental conditions and season on disease severity
Map distribution and impact (disease score for at least 20 plants per site and banana variety) of target pest and pathogen populations collected from surveys
Map distribution and impact (disease score for at least 20 plants per site and banana variety) of target pest and pathogen populations collected from visits on station and to regional test sites
Two samples from NARITA plants at Arusha have been received and were identified
Identification of Foc isolates from trial sites will continue for the next 2 years
0% variance
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.
50% variance
Reason: Regional trials were planted late and 2016/2017 was extremely dry. This affected plant establishment and symptom expression:
Mapping of nematode distribution in Tanzania is progressing while nematode sampling in Uganda is in progress
Reason: Mapping in Tanzania in progress. Sampling of nematodes in Uganda in progress
No data has been received on banana weevil from regional testing sites.
Reason: Data on weevils from regional testing sites not yet available since the plants have not yet been harvested

11. Leaf spot severity in low altitude (<1200 m a. s
Leaf spot severity in low altitude (<1200 m a.s.l), mid altitude ( ) and high altitude (>1500 m a.s.l) ranges in Uganda and Tanzania

12. Intermediate Outcomes
Outputs
Targets/ Milestones
Progress
Variance
YEAR 1
YEAR 2
YEAR 3
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
Evaluate resistance of 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
Trials being prepared
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.
0% variance
Evaluation of EAHB hybrids EAHB 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.

13. Sigatoka severity index computed based on severity per leaf as described

14. Damage due to banana weevils (Natural infestation) on Matooke genotypes at Kawanda
% total cross section damage
Motooke cultivar

15. Intermediate Outcomes
Outputs
Targets/ Milestones
Progress
Variance
YEAR 1
YEAR 2
YEAR 3
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
Evaluate resistance of 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
Trials being prepared
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
Complete biannual visits on station to assess impact of target pest and disease species on Mchare diploids
Screening and field trials are being established at Kawanda in Uganda and Arusha in Tanzania.
90% variance
Reason: A delay in the multiplication and planting of Mshare diploids. Trials to be established in March and April 2017.
8 Mchares have been evaluated at least 3 times in Kawanda and samples collected.
0% variance
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
Reason: A delay in the multiplication and planting of Mshare diploids.

16. Intermediate Outcomes
Outputs
Targets/ Milestones
Progress
Variance
YEAR 1
YEAR 2
YEAR 3
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
Evaluate resistance of 27 NARITA hybrids under varied field conditions at 3 regional field test sites and determine influence of plant development, environmental conditions and season on disease severity caused by Fusarium wilt, Sigatoka, nematodes and weevils
Trials being prepared
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
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.
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 established in the screen house at the HORTI-Tengeru Arusha to screen 20 NARITA banana from mother trial site and two local checks (Ndizi ng’ombe and Mchare) for nematode resistance using multiple species screening method.
40% variance
Reason: Data collection has not yet commenced
The data on banana weevil infestation at regional testing not yet available
Reason: Weevil infestation assessment expected after the NARITAS have been harvested

17. Intermediate Outcomes
Outputs
Targets/ Milestones
Progress
Variance
YEAR 1
YEAR 2
YEAR 3
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
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
MSc student recruited to characterize and partition resistance to nematodes and weevils in Musa
Existing biossays for Foc, Mycosphaerella spp., nematodes and weevils catalogued
Develop test runs to assess survival of banana material in vitro using different methods and to ensure suitability across varieties
Test suitability of in vitro methods across target pests and pathogens for at least 10 selected banana varieties
Bioassays for Foc, Mycosphaerella spp., nematodes and weevils adapted/ developed for high throughput screening.
Challenge selected banana varieties with representative populations of target pests and pathogens (from surveys) and document results
Factors determining resistance to target pests and diseases in selected cultivars determined (such as stomatal characteristics and epicutular wax for Mycosphaerella spp.)
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 mM
70% variance
Reason: The 19 cultivars from ITC arrived in March They will now be multiplied, and it is expected that the trial will be complete by June 2018.
Field trial with selected varieties was established in Sendusu in December 2016 to determine components of resistance. Screen house experiments established with suckers to optimise inoculum type and level will be inoculated in May.
50% variance
Reason: There was a delay in getting tissue culture plants
Multiple species screening method for nematode resistance has been adopted and is in use in the screen house experiment
0% variance
A field trial of 10 diploid bananas, a control 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)

18. Mean percentage suppression of Fusarium oxysporum f. sp
Mean percentage suppression of Fusarium oxysporum f. sp. cubense by plant phenolic compounds.

19. Intermediate Outcomes
Outputs
Targets/ Milestones
Progress
Variance
YEAR 1
YEAR 2
YEAR 3
Rapid and precise screening methods for Fusarium wilt, Sigatoka, nematodes and weevils developed for use in breeding programs
Compare the reliability of young plant resistance testing compared to whole plant testing in field for evaluations against Fusarium wilt, Sigatoka, nematodes and weevils
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
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 virulence testing are under tissue culture multiplication
Reason: Delay in getting tissue culture plants.
Screen house experiment for nematodes screening is progressing
60% variance
Reason: Field experiment need to be established
Established a field screening trial for resistance to banana weevils using three resistant cultivars; three susceptible cultivars; and four NARITA hybrids 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.
Reason: Trials need to be planted in April and May 2017

20. Intermediate Outcomes
Outputs
Targets/ Milestones
Progress
Variance
YEAR 1
YEAR 2
YEAR 3
Train staff in banana disease evaluation, resistance screening and pathogen and pest identification with focus on Fusarium wilt, Sigatoka, nematodes and weevils
Train staff on survey and culturing of Fusarium wilt, Sigatoka, nematodes and weevils
Stellenbosch univ (Fusarium), Sigatoka (IITA), weevil (NARO), nematodes (ARI-Tengeru).
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
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.
0% variance
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.

21. Training enumerators on collecting sigatoka data and harmonize disease screening protocols.

22. 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.

23. 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 GA3 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 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.

24. 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.

25. 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.

26. Summary
Significant progress has been made to get the project back on schedule. This include collections of pests and pathogens at all locations
Problems with Sigatoka isolations and identifications had been resolved.
Problems encountered with quarantine regulations for movement of weevils and nematodes across country borders were resolved.
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.
Major delays were due to tissue culture problems and the availability of planting materials. This has now been largely resolved.
Communication within WP2 has been significantly improved, but intergroup collaboration still needs improvement.
Several training sessions were presented and attended in the past year, with good outcomes to WP2 and the entire breeding project.