Fusarium Wilt Disease of Chickpea in Sudan: Incidence, Pathogen Genetic Variability and Screening for Resistance
Chickpea (Cice rarietinum L) is a cool season food legume and an important cash crop in the Sudan. The crop was traditionally grown on residual moisture of the flood in northern Sudan and recently it is introduced to the central parts of the country as an irrigated crop. Fusarium wilt disease caused by the fungus Fusarium oxysporum. f. sp. ciceris (Foc) is one of the most important vascular wilt diseases of the crop in Sudan, which affects its production. A survey was conducted in seasons 2010/2011 and 2011/2012 in different chickpea production areas to assess the incidence of the disease, its correlation to farmers’ cultural practices and to identify the genetic diversity of the pathogen population in Sudan. In this regard, 170 samples of chickpea wilted plants were collected from six States in the country (Northern State, River Nile State, Khartoum State, Gezira State, Sennar State and Kassala State). Information about the cultural practices done by farmers during the growing seasons, GPS data and samples of chickpea wilted plants were collected in each location. The survey data were analyzed using SPSS 16.0 Program and a map for the disease was madeusing GPS data. The pathogen was isolated from collected samples and purified in single spore cultures in vitro and subjected to cultural, morphological and pathogenicity studies. To identify the genetic diversity among the Sudanese Foc isolates, 90 distinct isolates were studied including 76 isolates obtained from different chickpea growing regions of Sudan and 14 isolates obtained from Syria and Lebanon for comparison. The DNA of all the isolates were extracted and subjected to molecular characterization by three types of molecular markers. The PCR analysis were carried out using four Random Amplified Polymorphic DNA (RAPD) primers, three Simple Sequence Repeats (SSR) primer pairs, 5 Sequence Characterized Amplified Region primers (SCAR) and 2 specific Foc identification primer sets. Gezira State gained the highest chickpea surveyed area and the most popular chickpea xv variety grown was the small kabuli "Baladi" variety. This variety occupied about 64% of the total surveyed area and was the most susceptible variety to the disease similar to Shendi and Jebel Marra cultivars (20-25% disease incidence). The incidence of the disease in the heavy clay soils of central and southern Gezira State (21 and 27%) was higher than that in the light clay and sandy clay soils (11-12%). The area under chickpea preceded by cereals in the rotation accounted for 40% of the total surveyed area followed by fallow (15%) and less than 10% for vegetables, and monocropping. Disease incidence was the highest in the monocropping system (42%), whereas in chickpea fields preceded by cereals, cotton, fallow and vegetables, it was 22, 20, 12 and 8%, respectively. Sowing date predominantly used by farmers was mid November. Early sowings during this period were subjected to high wilt disease incidence (31%) as compared to late December sowings that exhibited only 5% disease incidence.The seeding rate varied from farmer to farmer and the disease reached 50% in fields grown with more than 80 kg/ha. More than 40% of the farmers watered their crops at 2 weeks interval. The crop subjected to shorter (weekly) watering intervals had lower disease incidence (16%) than that subjected to longer intervals (22 and 24%). Around 54% of the farmers did not apply any kind of fertilizers to chickpea and 45% apply starter dose of Urea (2N/ha) and very few farmers added foliar fertilizers. Addition of fertilizers had no significant effect on disease incidence. Testing of 25 Foc isolates collected from Gezira, Khartoum, Sennar, Kassala and River Nile States with a set of chickpea differential varieties resulted in resistant reaction of these differentials to the tested isolates. This reaction is similar to the reaction of these differentials to race 0 in previous studies. When the isolates were cultured on PDA media, distinct variations were noted among the isolates with respect to colony diameter, texture and colony color, measurements of macro and micro conidia, abundance and absence of chlamydospores. The size of microconidia was in a range of 3.75-12.9 x 2.1- 3.4 μm. The longest and the shortest microconidia xvi were observed in isolates of central and southern Gezira State, respectively. There were no significant variations among micrconidia length and width of all the isolates. Generally, macroconidia size of all isolates was in a range of 17.5- 42.5 x 2.5-6.25 μm and the longest macroconidia was observed in Altalha isolate of central Gezira, whereas the shortest was observed in Alburgaig (Northern State) and Hudeiba (River Nile State) isolates. The widest macroconidia was observed in Daressalm area of central Gezira. Chlamydospores were detected only in isolates from central Gezira, Sennar, Hudeiba and Shambat sick plots and Syrian isolates. Four RAPD and three SSR primers were used to assess genetic diversity among 90 isolates collected from six chickpea growing states in Sudan. They were compared to Syrian and Lebanese isolates with known race identity. It is apparent that all the isolates are Fusarium oxysporum f. sp. ciceris but they are different from the Syrian and Lebanese isolates. Based on the coefficient of similarity, the isolates were grouped into two different major clusters and seven sub clusters in the dendrogram. The minimum dissimilarity value between the isolates was 0.1 and the maximum value was 1. These clusters differentiated the Foc isolates of Sudan based on the races nomenclature to race 0, 2 and unidentified race. The cluster analysis clearly distinguished the unidentified Foc strains obtained from central Gezira State from the other Foc isolates. Race 0 is widely distributed in central Sudan, while the unidentified race is restricted to Gezira State. Race 2 is distributed in Northern State, River Nile State and northern part of Gezira State. The Syrian and Lebanese Foc isolates which have been included for comparison were sub clustered separately which coincided with their expected races 1B/C and 6, respectively. Twenty chickpea germplasm were screened against the three identified races. The cultivar Hawata showed resistant reaction to the three tested Foc races, while Shendi and Jebel Marra cultivars showed susceptible reaction to race 0 and highly susceptible reaction to the other two races. The other genotypes showed susceptible reactions to the unidentified race and xvii variable reactions to races 2 and 0.It is evident from this study thatthe specific molecular markers used are the most rapid, reliableand effective tools in characterization and race identification of Fusarium oxysporum f. sp. ciceris. In addition, these findings will contribute to not only design and develop effective management strategies for chickpea wilt disease but will also help the breeders to design effective disease resistance breeding programs in chickpea.