Research work performed during this study was aimed to elucidate the role of virulence factor in breeding resistance to Ascochyta rabiei. During this study nine isolates of A. rabiei which is a causative agent of blight of chickpea, were screened for the presence of phytotoxic activity. Optimum conditions like medium, pH, temperature etc. were investigated for the growth of A. rabiei. Biotoxicity of fungus free culture filtrate of A. rabiei was confirmed by cells, nodes/internodes, detached leaves and root growth inhibition bioassays. Further investigations indicated the presence of phytotoxic compounds associated with fungal pathogenesis in chickpea blight. It was observed that the synthesis of phytotoxic metabolite(s) was induced in the presence of unknown endogenous factors present in the aqueous extract of chickpea seeds. In order to detect the phytotoxic compound(s) during growth of A. rabiei in Czapek Dox medium supplemented with chickpea seed extract, the toxins were isolated from the culture filtrate by organic extraction, solid phase extraction and HPLC and identified as solanapyrones A and C. Further studies were carried out to establish a pattern of production of these phytotoxin(s) in 12-day old fungus free culture filtrates of nine different fungal isolates. They were grown in still culture on Czapek Dox liquid medium supplemented with aqueous extracts of the chickpea seeds of one of two cultivars, a desi (6153) or a kabuli (Cypressa). Toxicity of the culture filtrates from both media were assayed with the cells isolated from the leaves of both cultivars and concentrations of the phytotoxins, solanapyrone A and solanapyrone C, in the filtrates were determined by high performance liquid chromatography (HPLC). For eight of the fungal isolates, toxicity and solanapyrone concentration were correlated; the value of r varies from 0.383 to 0.859 according to the cultivar used in the medium and assays. But in the culture filtrate of one isolate, there was no detectable level of solanapyrones but was showing 15 to 20 times more toxicity as compared to other isolates. The compound was isolated and found to be cytochalasin D by mass spectrometry and NMR. Solanapyrone C and cytochalasin D were also detected from the naturally infected chickpea plants.
To find out the role of isolated phytotoxic compounds in blight of chickpea, the content of phytotoxic compounds in artificially infected chickpea plants with different fungal isolates of A. rabiei has been determined. The severity of disease was recorded using a scale ranging from 1 to 9 (Grawel, 1981) having five defined categories of severity after 12 days. Infected plant materials were also processed through minicolumn for the quantification of phytotoxic compounds by HPLC during pathogenesis.
The studies indicate that considerable variability exists with isolates of A. rabiei for pathogenicity and virulence. The lowest grade of disease severity on 1-9 was 3 and the highest 9. Thus no cultivar showed complete resistance. Maximum phytotoxic compounds were detected on the 12th day of infection, Toxin production varied in both cultivars of chickpea infected with different isolates of A. rabiei varying in virulence. Cytochalasin D was recovered in high levels followed by solanapyrones B + C and solanapyrone A. However, the correlation between the titers of solanapyrones A, B + C and cytochalasin D synthesized by the various fungal isolates upon plant infection and the severity of the disease as determined by the size of leaf and stem lesion was calculated as 0.081, 0.144 and 0.718 respectively on disease susceptible chickpea cv. 6153 while it was 0.055, 0.25 and 0.122 respectively on fungus resistant chickpea cv. C44. The results seem to negate a causative role of isolated phytotoxic compounds in the onset of fungal blight in chickpea.
For the determination of kinetics of phytotoxins production during in vitro growth of various isolates of A. rabiei varying in virulence, the culture filtrates were processed upto 24 days through minicolumn with the interval of 24 hours to resolve the contents of various phytotoxic compounds. Half of its volume was used for the quantification of phytotoxic compounds by HPLC and the other half was evaporated to residue to check the toxicityby cell bioassay.
Solanapyrones, A, B + C and cytochalasin D production varied among the nine isolates of A. rabiei, whereas fungal growth as evidenced by fungal dry weight and sporulation as observed under microscope proceeded normally. Insignificantly low levels of phytotoxic compounds were detected in the culture filtrate of isolates CAMB 7 and 8. In CAMB 8, only solanapyrone B + C were detectable at 2-3 ug/mg of the fungus. Whereas in other isolates production of the varying levels of phytotoxic compounds was observed during in vitro growth and cytochalasin D was observed only in isolates CAMB 5 and CAMB 10. These results indicate CAMB 7 to be a Tox isolate at the present detection limits whereas CAMP 8 produces only solanapyrone B + C but not solanapyrone A. Analysis of genetic diversity by fingerprinting of A. rabiei isolates was one of the measurable objective of the programme. Nine isolates of A. rabiei were characterized by DNA fingerprinting with synthetic non radioactive labeled probes complimentary to simple repetitive sequences namely. (GATA)4, (GACA)4, (GTG)5, (CAA)5, (CA)8, (GGAT)4,Genome of A rabiei was studied and confirmed various isolates of A. rabiei (Previously categorized according to the disease severity and toxin production). All sequence motifs were found to be present in the A. rabiei genome differ in organization and abundance. The extent of polymorphism observed and informativity obtained are strongly dependent on the probe as well as on the restriction enzyme used. Taql and Hinfl are found to be most suitable for oligonucleotide fingerprinting of A. rabiei genome as compared of EcoRI. Among six probe, (GATA)4 was the most informative, occurred less frequently and appeared to be concentrated at one or two predominant loci. In contrast, the motifs (GACA)4, (GTG)5, (CAA)5, (CA)8 and (GGAT)4 fingerprints were more complex and pattern created by (GACA)4 and (GTG)5 probes were district but monomorphic. Seven isolates, out of nine, were similar with one another but different from the rest of the isolates when hybridized with (CA)8 and (GGAT)4.