ISOLATES OF ASCOCHYTA RABIEI
6.1 IN TR O D U C TIO N
Isolates o i Ascochyta rabiei produced solanapyrones A, B and C in three
liq u id media (as seen in chapter 3) and the solanapyrones caused death o f
isolated chickpea cells (as seen in chapter 5). Moreover, the solanapyrones
affected different cultivars to different extents in the single cell assay. This
suggests that the toxins may contribute to the developm ent o f disease symptoms. To test the relationship of solanapyrone production and virulence, isolates were grown in Czapek Dox liquid media supplemented w ith the defined metal
cation solution and grown over a period of 2 1 days to measure the quantity of
solanapyrones produced. This medium was chosen to compare toxin production by isolates because o f the reproducibility o f the results as found in chapter 3. Virulence of isolates was tested by inoculating three cultivars o f chickpea and using the Linear Infection Index (III) to measure the a b ility o f the fungus to infect the plant (Riahi et a/., 1990). In this technique the number o f lesions (ML) and
average lesion length (ALL) produced on the stem was measured. ALL was
obtained by adding the length of all lesions on each stem (TLL) and d iv id in g by the number o f lesions on the stem. Stem length (SL) was calculated from the base o f the plant to the shoot apex (including branches). From these measurements a
Linear Infection Index (Lll) was calculated as LI I = NL X ALL / SL expressed as
a percentage.
This index best described the reaction o f chickpea seedlings to A. rabiei
(Riahi et a!., 1990) and being based on quantitative measurements of the disease
expression, was found to separate resistant and susceptible plants better than the 1-9 scale developed by ICARDA (Singh et a/., 1981) w hich uses mostly visual disease assessment.
In this chapter the hypothesis that the virulence o f isolates is related to solanapyrone production is tested.
6.2 MATERIALS A N D METHODS
6.2.1 Toxin production by isolates of A. rabiei
6.2.1.1 Liquid media
Czapek Dox liquid media supplemented w ith the defined metal cation solution (ZnSO^ concentration of 50mg/l) were prepared as in section 3.2.1.
6.2.1.2 Analysis of culture filtrates
A ll culture filtrates were harvested (as in section 3.2.5.) and the
solanapyrones were extracted by solid phase (section 3.2.6.1.). A ll culture
filtrates, washes and purified samples were analyzed by HPLC (section 3.2.7.3.) and the single cell assay (as in section 5.2.).
6.2.1.3 Solanapyrone degradation
Isolate 6 K was grown on Czapek Dox liquid media supplemented w ith the
defined metal cation solution (ZnSO^ concentration of 50mg/l) section 3.2.1. After 30 days the culture filtrate was harvested (section 3.2.5.) and divided into
15 portions.
The first three portions were analyzed for solanapyrones (section 3.2.7.3.). To the other portions a solution containing solanapyrone A = 0.32m M , solanapyrone B = 0.40m M and solanapyrone C = 0.55m M was added.
Samples were filte r sterilised through sterile 0.2/ym (Whatman) filte r paper
and incubated at 20°C in the dark for 1 hour, 5 hours, 24 hours and 72 hours.
The samples were analyzed for solanapyrones (Section 3.2.7.3.).
6.2.2 Testing of virulence
6.2.2.1 Plants
Cultivars ILC-3279, ILC-482 and Am doun were used in the tests. Chickpea
seeds were soaked in distilled water at room temperature for 1 2 hours and grown
as in section 2 .2 .1 .
Plants were inoculated 14 days after germination (section 2.2.3.) and incubated in growth chambers at a temperature of 20°C, w ith a photoperiod of
16hrs daylight per day at an overall light intensity of 2 0 0 //m ol m V at plant
height.
6.2.2.2 Disease evaluation
Disease reaction was evaluated by the Linear Infection Index o f Riahi et a/. (1990). The num ber of infected plants at the beginning of the experiments was zero w ith an observed frequency of 100% at LH% = 0, i.e. no lesions. O ver the weeks, disease symptoms developed and the observed frequency of infected plants for each Lll% increased. When the total lesion length covered 11 % of the stem length or Lll% = 11 or more, the plant was deemed susceptible to the fungal isolate. Infected plants w ith Lll% values greater than 11 are not included
in the histograms as they were usually dying or dead.
Controls were run in conjunction w ith the treatments. The first control (negative control) consisted of uninoculated plants of the 3 cultivars and the second control (positive control) consisted of the 3 cultivars inoculated w ith a
spore suspension of the most virulent isolate 6 K (PUT7).
At the end of the experiments the sand and plants were autoclaved at 121°C for 30 minutes and the pots were washed in a 5% sodium hypochlorite solution, then w iped w ith ethanol.
6.2.3 Mating of isolates
A ll isolates of A. rabiei collected and single spored were crossed against
each other (Table 4). Pycnidiospore suspensions of the various A. rabiei isolates
were made up to a concentration of 1 X 10^ and mixed together aseptically.
6.2.3.1 Plant material
Plants of the cultivar Am doun were grown as in section 2.2.1. and were harvested at various stages in the life cycle (Appendix 5). The plant material was surfaced sterilized w ith a 5% sodium hypochlorite solution for 5 minutes or w ith
propylene oxide for 1 minute, then washed three times w ith sterilized distilled
w ater. Plant pieces were dried w ith sterile tissue before inoculation.
6.2.3.2 Inoculation of the plant material
Spore suspensions were mixed together in a sterile beaker. The plant pieces were placed in the spore solution for 10 - 30 minutes and transferred to either sterilized moist tissue paper (w ithin sterile plastic bags and sealed) or placed in sterile petri dishes (containing 5ml of sterile distilled water and 5 pieces o f sterilized W hatman no.1 filter paper). They were incubated at 4°C, 10°C, 15°C
fo r 2 months.
6.2.3.3 inoculation of growing plants
Plants were grown as in section 2.2.1. and inoculated w ith a spore
suspension containing tw o isolates of A. rabiei (as in section 2.2.3.) and left to
grow w ith in muslin cages at a temperature of 10°C - 19°C for 2 months.
A few plants from each experiment were harvested at w eekly intervals and