Constraint #1: Insufficient Natural Resource Management and Production Technologies

Research area: Pest and disease control through genetic and agronomic means

Background: Pathogens and pests are a major constraint on bean production in East Africa. Pathogens of importance are angular leafspot (ALS) common bacterial blight (CBB) and bean common mosaic (BCMV) and bean common mosaic necrosis (BCMNV) potyviruses. Nematodes constitute an important, but often overlooked problem, particularly in the lowland areas. Several breeding lines have been developed at SUA, Malawi and the University of California-Davis (UCD) for improved cooking quality, nematode resistance, and BCMV, BCMNV, CBB and ALS resistance. Lines with improved resistance to ALS, BCMV and BCMNV are currently being evaluated in Malawi and at UCD. The nematode resistant lines were obtained by crossing unadapted nematode resistant lines with SUA advanced lines.

Common bacterial blight caused by Xanthomonas campestris pv. phaseoli is a major constraint for bean production in Tanzania and Malawi, as well as being a problem in the more humid bean-producing regions worldwide. In addition, the European Union (EU) requires testing for the absence of the CBB bacterium before bean imports are allowed into any of the EU countries. Most of the varieties grown by farmers are susceptible. Previous studies using the PCR technique suggest the possibility of co-evolution between strains and bean gene pools (Mesoamerican and the Andean gene pools). The correlation between gene pools and strains of the CBB pathogen needs further study to confirm such relationship. Information generated from these studies will assist breeders to efficiently deploy resistance genes to the CBB pathogen. The PCR laboratory established at SUA and Bunda can be used to diagnose the presence of CBB in a seedlot. This activity could be particularly valuable to seed producers who ship snap bean seed to the EU.

The activities under this constraint are directed toward development of bean varieties that have high, stable yields, multiple disease resistance, and are fast cooking. Two varieties in Tanzania were developed and released with farmer input into the selection process. SUA 90 and Rojo are BCMV and rust resistant, generally stand up well to other fungal and bacterial diseases, and are adapted to low elevation growing environments. In Malawi, Kalima has been released with four MT produced by Bunda in FY97-98. Two NGOs and the Ministry of Agriculture have used this seed for further increase and distribution. Studies are under way to document impact at the farm level.

Socioeconomic surveys indicated that important types are Red kidney (Chimbamba), Red speckled on cream (Nanyati), Beige kidney (Nasaka), and White kidney and navy (Kayera), which are preferred across the country. Kaulesi type is preferred in the South and Nyauzembe in the north. As a result these types were used in crosses with resistant sources for ALS and BCMV from CIAT. Large-seeded adapted types were used in backcrosses. Progenies are being evaluated in Malawi and some should be released in two years.

Activity #1: Promote PCR capabilities (develop DNA protocols, screen RAPD primers; identify pathogens, develop diagnostic capabilities)

U.S. researchers: Gilbertson, Temple
HC researchers: Mabagala, Bokosi
Methodology: The PCR laboratory and capability will be promoted by distributing flyers to potential national, national agricultural research institutions, seed producers and graduate students, importers, exporters, etc. in the country and in the region in general. The intent is to develop fee-based research and diagnostic activities that will institutionalize the PCR laboratories.

In addition to flyers, visits to seed producers and exporters, especially in northern Tanzania (Moshi/Arusha), will be conducted to discuss with them and explain more of the need for testing seed using the SUA based PCR laboratory. The primary focus at SUA will be on establishing testing services for CBB and other seed-borne pathogens that would facilitate seed export.

We would also continue development of PCR capacity and facilitate training by involving undergraduate and graduate students.

PCR-based research will continue identification and characterization of CBB, halo blight and ALS pathogens, and facilitate co-evolution studies by determining gene pools and CBB isolates associated with them.

Screening of advanced bean breeding lines will be done using the collected isolates.

Anticipated results of activity: The use of PCR will bring scientists in Tanzania and Malawi closer to the cutting edge of research, and will support seed testing activities in the countries, which will establish sustainability for the PCR laboratory in the future. The co-evolution studies will guide breeders in the development of resistant varieties.

Anticipated impact(s) to which this activity will contribute:

• Cleaner seed stocks, higher quality seed will be available to farmers and for export
• Improved detection capability of plant pathogens will be established to facilitate development and production of pathogen-free seed for on-farm seed producers/importers/exporters
• A base of scientists capable of using PCR will be developed

Progress During Past Year (Tanzania): Visits were made to the National Germplasm Conservation Center (TPRI), Ilonga Agricultural Research Institute, Rotian Seed Company Ltd, Manyara Seed Estate, Bulka Estates, Pop Vriend, Asian Vegetable Research and Development Center (AVRDC) African Region-Arusha, Agricultural Programme Sector support (ASPS) – Ministry of Agriculture and Cooperatives, Dar es Salaam and Agricultural Research Institute Selian. The purpose of the visits was to promote the use of the PCR lab by creating awareness to potential users for diagnostic activities. Discussions were conducted with managers of bean seed growing companies on the advantages of using the SUA-based PCR laboratory for testing their seed before export, as compared to mailing the seed for testing in Europe or South Africa. Generally, bean seed growers contacted indicated that sending seed to Europe for testing was very expensive and time consuming. Therefore, they were very much interested in using the SUA PCR laboratory in that testing bean seed for CBB and HB at SUA would reduce mailing costs, and the time lag for results. Most companies requested that sample testing for seed-borne inoculum should remain confidential and that results should not bear the name of the laboratory. Some bean seed companies indicated also that they would like to use the PCR lab at SUA to test their bean seed stocks for CBB and HB before planting during the year 2001. Such testing will ensure that they will begin with clean seed stocks.

A total of 500 flyers to be used for promotion of the PCR laboratory were prepared and are being printed. These flyers will soon be distributed to potential customers who will be using the PCR laboratory for testing seed for CBB and HB.

The development of the PCR laboratory continued by importing Taq Polymerase enzyme from Denmark using leveraged funds from DANIDA since the previous stock had expired. Furthermore, one Ph.D. student from Nigeria who is working on Tomato Yellow leaf curl virus (TYLCV) is currently using the PCR laboratory under the supervision of Dr. R. B. Mabagala with some assistance from Dr. R. Gilbertson. It is anticipated that on his return to Nigeria, the student will be well equipped with PCR techniques and procedures. In addition, Dr. James Bokosi from Bunda College and Dr. S. B. Mathur from Denmark visited the PCR laboratory to discuss various issues related to the use of the PCR laboratory for testing on-farm produced seed.

Malawi: The PCR remains underutilized. Students taking molecular biology are exposed to the equipment to learn about its operations and capabilities. We will however, need some fresh orders on some supplies. Plans are underway to sensitize the seed industry, agriculture research institutions and others on the aim of developing a fee-based research and diagnostic services.

UC-Davis: Detection of CBB bacteria in seed samples: During 2000, PCR was used to monitor a common bacterial blight (CBB) epidemic on bean seed crops in California. In January 2000, samples of 14 seed lots were sent to our laboratory to assay for CBB bacteria. This seed had been produced in Wisconsin where CBB was present in the fields. Ultimately, some of this seed was to be planted in California. The seed was checked for the presence of Xanthomonas campestris pv. phaseoli (Xcp) and X.c. pv. phaseoli var. fuscans (Xcpf) with a standard seed wash assay and plating on MXP medium. Colonies were counted after 4-5 days. The seed from all lots was heavily infested with Xcp (up to 16.9x10⁷ cfu/g seed). Lot 14 appeared to have a lower level of contamination.

We then designed an experiment to determine where in or on the seed bacterial inoculum was located. Seeds from contaminated lots were bulked and mixed thoroughly. Half the seeds were surface sterilized with 5% Physan 20 or left untreated. Half of the seeds from each treatment were either left intact or were crushed. The experiment was repeated twice. The four seed lots were washed and bacteria were recovered as described above. When the bulked sample was constituted and thoroughly mixed, the seeds contained an average of 1.8x10⁷ cfu/g of Xcp (Table 1). Crushed seed had even more bacteria (mean of 3.1x107 cfu/g). Physan 20 eliminated bacteria from the surface of the bean seed, however, crushed Physan treated seeds had high populations of bacteria (mean of 2.5x10⁷ cfu/g). This experiment confirms that significant CBB contamination is beneath the seed coat.

Some of this seed was planted in fields in California in 2000. Several seed lots were grown under sprinkler, while one was grown under furrow irrigation. CBB symptoms were observed in the sprinkler-irrigated fields (Fields 2, 3, and 4), but no symptoms were observed in the furrow irrigated field (Field 1). Field 2 also had possible bacterial brown spot (P. syringae pv. syringae) symptoms. No blight bacteria were isolated from Field 1, but was detected in the other three fields. The presence of Xcp was verified based on colony appearance and amplification with Xcp/Xcpf primers.

We employed the rep-PCR to determine if the DNA fingerprint from strains recovered from the field were similar those isolated from the seed. The fingerprints were indistinguishable supporting the conclusion that the inoculum source was the seed.

Current Status of Research: The PCR laboratories at SUA and Bunda were initiated in 1998. The use of the PCR facilities for seed testing of on-farm produced seed and bean seed from large producers, exporters and importers has been promoted through visits to potential customers and preparation of flyers. These flyers will be distributed soon. Bean seed producers and exporters have accepted to use the PCR facility at SUA to reduce the cost of mailing the samples abroad for HB & CBB testing. SUA is now working out the cost element involved in testing such samples so that appropriate fee charges can be established for each sample testing. The PCR laboratory needs to be further supported before it can generate revolving funds to run its activities and to strengthen the laboratory further.

Documented impact: Training in the area of biotechnology using the PCR laboratory at SUA has been initiated. A Ph.D. student from Nigeria is currently conducting his research using the PCR laboratory under the supervision of Robert Mabagala. PCR provides a major tool for detection and characterization of CBB, and was used to document the occurrence of the disease in seed fields grown in California. A related activity from past years has been testing and identification of bean viruses in California. This program is mature enough to have created some impact. The virus detection program has been widely acclaimed in California as (1) helping to reduce the incidence of BCMV in California seed, (2) raising overall awareness about bean-infecting viruses in California, and (3) providing an early warning system for the appearance of new bean-infecting viruses in California. This is further evidenced by the leveraged funds we have obtained from CCIA to partially fund this program and the fact that CCIA has decided to have our laboratory as the center for the virus detection program.

Activity #2: "Fingerprint" Kalima and Bokosi/Temple lines

U.S. researchers: Gepts, Myers, postdoctoral research associate
HC researchers: Mabagala, Nchimbi-Msolla, Bokosi
Methodology: Evaluate available DNA extraction protocols to identify most appropriate techniques for Malawi and Tanzania. At UCD and OSU, screen primers and develop PCR fingerprints.

DNA of bean lines from the Malawi and Tanzanian programs will be isolated and subjected to analysis using a number of different PCR primers to characterize, or establish a "fingerprint" for these lines. Number of primers evaluated will depend on the number needed to establish unique identity. Work will be carried out first in the U.S. to establish the best protocols and set of primers for fingerprinting. Protocols will then be transferred to the Host Countries. Existing landraces, promising breeding lines and released varieties will be evaluated.

Anticipated results of activity: Identify protocols and primers for fingerprinting Malawi and Tanzanian advanced lines. Transfer of fingerprinting/cultivar identification technologies for use in the Malawi and Tanzania PCR laboratories. Establish fingerprints for newly released program materials to allow social scientists documenting dissemination to unequivocally determine the identity of bean lines found in the field–particularly important for varieties that cannot be distinguished from landraces based on seed type. As an example, one or more 'Kablanketi' type lines will be released in the next year or so. This type is one of the most popular in many parts of Tanzania and several varieties already exist. Determination of dissemination rates for an improved line would be difficult without a marker.

Anticipated impact(s):

• Technique can provide a quantitative tool to measure seed dissemination of improved varieties to farmers and through the markets. It is particularly useful where several varieties have the same seed type. Knowledge of germplasm organization, allowing more efficient choice of parents for crosses.

Major Changes: The UC-Davis program is investigating microsatellites for fingerprinting bean cultivars from Malawi, while the OSU program has focused on RAPDs. Microsatellites or simple sequence repeats (SSR) are short stretches of DNA, consisting of tandemly repeated nucleotide units (1-5 nucleotides long). They are usually very polymorphic due to the high level of variation in number of repeats. Differences in number of repeats is detected by running PCR products, amplified using a unique pair of primers flanking the microsatellites, on high-resolution gels. Compared with other markers, microsatellite markers have a number of advantages including: (1) they are codominant and PCR-based; (2) they are usually multi-allelic and hyper-variable; (3) they appear to be randomly and uniformly distributed throughout eukaryotic genomes; (4) they are accessible to other research laboratories via published primer sequences; and (5) they are highly reproducible.

Progress During Past Year: UC-Davis: Fifteen SSRs were recently characterized in beans (Yu et al., 2000). Those SSRs using 4 or more alleles have initially been used for fingerprinting. They are J04555 (PV-ctt001), U77935 (PV-gccacc001), X61293 (PV-at004), X74919 (PV-ct001), X80051 (PV-at007), and M99497 (VA-ag001). The East African bean genotypes being fingerprinted are: (1) Andean materials: 22-2 and 12-4, Rose Koko and Canadian Wonder from Malawi and Tanzania, respectively; (2) Mesoamerican materials: Masai Red from Tanzania and Namajengo, 6-5, and 1-1 from Malawi. White Kidney and Black Turtle Soup are U.S. Andean and Mesoamerican genotypes, respectively. DNA was isolated using a standard liquid nitrogen-mercaptoethanol-PVPP extraction protocol for obtaining large quantities of very pure DNA. The products were amplified by PCR, then fractionated on a polyacrylamide gel, and visualized by silver-staining. Fragments ranged in size from ~100 bp to about 500 bp. X80051 primers for the (AT)₁₂ simple sequence repeat resulted in a major fragment of about 500 bp across all six genotypes used. X61293 primers for the (AT)₁₈ simple sequence repeat produced many fragments with major ones of ~550 bp, 300 bp, 280 bp, and 250 bp and clear polymorphisms were identified among the six genotypes. U77935 primers for the (GCCACC)₅ simple sequence repeats produced fewer fragments (200-250 bp) with clear polymorphisms among the six genotypes. J04555 primers for the (CTT)₃(T) ₃(CTT)₆ simple sequence repeat revealed many polymorphic fragments among the six genotypes. X74919 primers for the (AT)₅ simple sequence repeat produced two major bands between 300 and 400 bp, with no polymorphisms. M99497 primers for (AG)₁₂(AGG)₂ produced three main fragments between 280 and 450 bp, with no polymorphisms. These results show that we can successfully use microsatellites to fingerprint bean genotypes particularly by using the primers that produced polymorphic fragments for different bean genotypes. We will later fingerprint Kalima, Nasaka, Sapelekedwa, Namajengo, Nyauzembe, Kayera, Nanyati and other parents in the Temple/Bokosi crossing block and also all resulting promising materials that are in advanced stages for release in Malawi.

OSU: Dr. Susan Nchimbi Msolla spent three months at OSU learning PCR techniques as applied to bean. She used 52 bean varieties and breeding lines from germplasm collections in Tanzania, Kenya, and Uganda. DNA was isolated using a fast (1 day) CTAB procedure that requires little plant tissue (<0.5 g) to produce adequate quantities of sufficiently pure DNA for PCR amplification. A RAPD protocol developed in the OSU laboratory for beans was used. Seven RAPD primers produced 12 reliable polymorphic bands that were used to characterize the bean accessions. Unweighted pair grouping by arithmetic means (UPGMA) and neighbor joining (NJ) were used to construct dendrograms based on shared markers among the lines (Figure 1). NJ showed closer agreement with known relationships among lines although the out-group (Lima bean) clustered with the small-seeded Mesoamerican group. A major split in the dendrogram identified Mesoamerican and Andean origins for the lines. The check varieties 'UI 111' pinto and 'Black Turtle' clustered with the Mesoamerican group. The snap bean variety 'Contender' clustered with the Andean group, but was distantly related to other Andean lines. Many of the types with the same name or seed class clustered together. For example, all 'Soya' (and 'Kablanketi') types clustered together although collected from different regions. Polymorphism within some landraces was detected. For example, two samples of 'Canadian Wonder' had different banding patterns. We also detected evidence of the release of improved varieties within certain market classes. Within the Rozikoko type, we observed two groups, one that clustered with the improved Rozikoko line 'Selian 94', and a second group that clustered with 'Canadian Wonder'. The latter Rozikoko types are most likely from the original landrace. 'Rozikoko mix' and 'SUA 90' had identical RAPD banding patterns. When we examined the seedlot, we determined that we had selected from mix of types in that seed lot a brown seeded type identical in appearance to 'SUA 90'. This seed lot was collected during our 1998 virus survey trip from Bungoma, Kenya. With relatively few RAPD markers, it appears possible to adequately characterize and fingerprint existing east African germplasm. The use of additional markers might allow finer discrimination among lines, but present results is adequate to be able to identify improved lines in landraces where seed appearance is identical. As such, fingerprinting has utility in ex post impact assessment to document the dissemination and spread of improved varieties from our breeding programs.

Current Status of Research: This activity began in 1999. Next steps are to extend the screen to additional African bean lines with focus on Malawi landraces and improved lines. Microsatellites will complement RAPDs in the detection of polymorphisms. Microsatellites should also be done on the Tanzanian materials, and RAPDs done on the Malawi lines. A sequencing gel apparatus will be needed to conduct microsatellite studies at Bunda.

Documented impact: Our bean breeder at SUA, Dr. S. Nchimbi-Msolla is now well acquainted with PCR-techniques after receiving three months training at OSU. She is currently characterizing bean genotypes at SUA. Detection of SUA 90 in a sample of beans purchased in Kenya illustrates the potential of this technique to quantitatively document varietal dissemination.