Abstract:

In Uganda, a major pathogen genus causing severe Phaseolus bean root rot, particularly in southwestern, is Pythium. But there is no information on the different Pythium species associated with bean root rots nor about impact of use of organic amendments on Pythium populations in field soils. This study was therefore, undertaken to a) identify Pythium species pathogenic to beans and map their distribution in major bean producing areas of Uganda, and b) develop a procedure of quantifying Pythium populations in field soils and c) to evaluate the impact of soil organic amendments on inoculum levels of Pythium spp. pathogenic on beans (Phaseolus vulgaris L). The first part of the study involved morphological, molecular and pathogenicity analyses of Pythium isolates collected from Kisoro and Kabale districts in southwestern Uganda and Mbale in the east. Molecular analyses using PCR-RFLP and sequencing techniques were used in the identification of Pythium species. Eleven Pythium species were found to be associated with bean root rots in Uganda. Apart from the traditional species, P. ultimum var ultimum, known to infect beans, other Pythium species recovered from infected beans were P. spinosum, P. torulosum, P. salpingophorum, P. vexans, P. dissotocum, P. nodosum, P. echinulatum, P. pachyaule, P. oligandrum and P. deliense. All isolates of P. ultimum var uitimum and P. spinosum caused severe root rot on susceptible bean varieties CAL 96 and K20. The other isolates belonging to P. torulosum, P. nodosum and P. pachycaule were also pathogenic to beans varieties CAL 96 and K20. Pathogenicity tests indicated significantly more disease (8 to 9 score) when highly pathogenic Pythium species were used to inoculate susceptible bean varieties CAL 96 and K20 compared to the highly tolerant variety RWR 719 (score of 2 to 5).Generally, morphological, molecular and pathogenicity analyses revealed that there is wide diversity within Pythium population in Uganda. The results obtained demonstrated that P. ultimum varultimums is responsible for the increased disease epidemics in Kabale and Kisoro districts. But, pathogenicity of P. spinosum, P. nondosum and P. pachycaule isolates suggest that these species also cause bean root rot under field conditions. Isolates of P. torulosum and P. salpingophorum were more variable in their pathogenicity, but the species could probably contribute to bean root rot as part of the complex of minor pathogens. Some PCR species-specific primers with good level of specificity to detect and differentiate P. salpingophorum, P. torulosum, P. spinosum, P. nodosum and P. ultimum, were developed during the study. The limitation of the primers was their failure to differentiate some p articular Pythium species with variation in pathogenicity, a s was the case with P torulosum and P. salpingophorum. Further molecular analysis of other regions of the genome is required to locate genes responsible for the variation and later design primers specific to pathogenic forms of P. torulosum and P. salpingophorum. The second part of the study involved quantification of inoculum levels of Pythium spp. pathogenic to beans, using the dilution-plating method with a selective media, in combination with molecular PCR-RFLP identification procedures. Soils infested at ratios of 1:5 and 1:10 consistently produced large numbers of colonies when 1 ml of suspension from the 50- or 100-ml volumes was plated out. It was difficult to count the colonies, as in the second day they were merged. Further dilution to 200ml produced countable colonies (15 per plate; equivalent about 1500 cfu.g’1). While soils infested with Pythium at ratios of 1:15 and 1:20 v/v consistently produced fewer colonies (about 2 per plate). The dilution plating procedure developed to estimate Pythium populations was plating 0.7ml (instead of 1 ml) of a suspension of two grams soil in 100ml of sterile water. The numbers of colonies growing on each plate was about seven (equivalent to 500 cfu.g'1). Two to three colony types were observed on plates containing samples from the naturally infested field soil from Kicumbi, suggesting the different possible Pythium species occurring in field soils. The identification using molecular techniques still differentiated the colonies into groups. The third part of the study assessed the impact of organic amendment (farmyard manure and Calliandra') on Pythium populations in field soils. Farmyard manure and Calliandra significantly increased Pythium populations in both screen house and field experiment, by a range of 200 to 700 colony forming units in the field trial and up to 1200 in the screen house relative to the control. Interestingly, Calliandra increased the frequency of P. salpingophorum by two times but reduced that of P. ultimum. On the other hand, farmyard manure increased frequencies of P. ultimum by about one and half but reduced those of P. salpingophorum. The mechanism by which the frequencies of particular Pythium species are reduced or increased is not well understood and should be investigated. Despite the increases in Pythium populations in field soils, amendments resulted into increased dry matter and bean grain yield. It is postulated that farmyard manure and Calliandra may be able to compensate for the negative impact of increased disease by causing a larger increase in soil fertility, thereby increasing yield. Further, both farmyard manure and Calliandra reduced Pythium population level in the follow up crop by about 50%. Then, they could be used as part of a series of management practices to reduce the impact of Pythium root rot on beans in a long run. This study has therefore, a) characterized the Pythium species responsible for bean root rots in southwestern Uganda, b) developed Pythium specific primers for detecting five Pythium species in both plants and soil c) developed a procedure for quantifying Pythium inoculum levels in the soil, and d) demonstrated the beneficial effect of organic amendments for reducing the impact Pythium levels in the soil. This knowledge will be useful for future development of bean varieties in the Great Lakes region of eastern Africa.