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
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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
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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
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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.