Table 1.
Primer sequences specific to Fusarium oxysporum f.sp. ciceri 1.5-kb sequence characterized amplified region (SCAR) (GenBank accession no. AF492451) used for quantification of the pathogen in chickpea roots using qRT-PCR.
Table 2.
Primer sequences of the virulence related genes and EF1α (used as a reference gene) used for qRT-PCR.
Table 3.
Phenotypic characterization of eGFP transformants of Foc 2.
Fig 1.
Early stages of chickpea root colonization by Foc 2 marked with eGFP in susceptible (JG62) cultivar by Confocal Laser Scanning Microscopy.
A. Uniform expression of eGFP in hyphae and spores of transformed isolate D4. B. Germinating conidium with primary mycelium in contact with root apex at 24 hpi. C-D. Initial hyphal colonization at lower root zone at 2 dpi. E. Intermediate root zone showing hyphal colonization extending from epidermis to cortical cells at 2 dpi. F-G. Vascular region of root getting colonized at 3 dpi. H. Fungal colonization in cortex region of DVI.
Fig 2.
Temporal pattern of colonization of Foc 2 in both susceptible (JG62) and resistant (Digvijay) cultivars of chickpea.
R- Root, S- Shoot, LR- Lower Root, MR- Middle Root, UR- Upper Root. A. (1–3 dpi) Surface colonization in both susceptible and resistant cultivars. B. (4–6 dpi) Transverse sections (TS) of root depicting entry of fungus in vascular bundle only in JGI. C. (8 dpi) Transverse sections of lower and middle roots showing increasing amount of fungus in vasculature of JGI while only few fungal mass seen in DVI only in LR. D. (10–12 dpi) Transverse sections of all lower, middle, upper roots of JGI reveal higher fungal mass in vascular tissue with few fungal mycelia reaching stem also. However, little fungal mass can be seen till cortex region of TS of LR in DVI with remaining TSs of MR, UR and Stem are clear.
Fig 3.
Temporal pattern of colonization of Foc 2 in both susceptible (JG62) and resistant (Digvijay) cultivars of chickpea.
R- Root, S- Shoot, LR- Lower Root, MR- Middle Root, UR- Upper Root. A. (14 dpi) Transverse section of root and shoot reveals still higher fungal mass in JGI while nearly absence of fungus in DVI.(slight fluorescence marked in the figure). B. (18 dpi) Transverse section of root and shoot showing vasculature completely flooded with fungal mass in JGI and very few fungal hyphae in DVI. C. (25 dpi) Transverse section of root and shoot reveal distortion of vasculature because of heavy fungal colonization in JGI whereas plant tissues are near normal in DVI with little fungus here and there.
Fig 4.
Late stages of chickpea root and shoot colonization by Fusarium oxysporum f.sp. ciceri race 2 marked with eGFP in susceptible (JG62) and resistant (Digvijay) cultivars at 28 DPI.
A. Heavy colonization of root of susceptible (JG62) plant in longitudinal section. B. Cross section of root of susceptible (JG62) plant showing complete colonization of fungus in cortex (C) as well as xylem vessels (X) and deformation of root architecture. C. Longitudinal section of stem of susceptible (JG62) plant with the extensive presence of conidia as well as mycelia of fungus. D-E. Cross and longitudinal sections of root of resistant plant (Digvijay) with absence of any fungus and normal architecture of roots. F-G. Longitudinal and cross section of stem of resistant (Digvijay) plant without any fungal infection.
Fig 5.
Proportion of Foc 2 DNA in whole roots of chickpea cultivars, JG62 (susceptible) and Digvijay (resistant), at different time-points after inoculation.
Amount of pathogen DNA was estimated using quantitative polymerase chain reaction (qPCR). Bars with asterisk (*) indicate level of significance of the amount of pathogen between JGI and DVI as per T-test performed between the two groups. ‘*’- P<0.05; ‘**’- P<0.01; ‘***’-P<0.001.
Fig 6.
Proportion of Foc 2 DNA in fractions of roots and shoots from the inoculated root tip of (A) susceptible (JG62) and (B) resistant (Digvijay) chickpea cultivars at various time-points after inoculation. In case of JG62, 28 DPI root fractions could not be collected as the plant was completely wilted. Bars with asterisk (*) indicate level of significance of the amount of pathogen between JGI and DVI as per T-test performed between the two groups. ‘*’- P<0.05; ‘**’- P<0.01; ‘***’-P<0.001.
Fig 7.
Expression patterns of eight fungal virulence related genes in JGI and DVI at eight different time-points using quantitative reverse transcriptase PCR.
Bars with asterisk (*) indicate level of significance of the expression of particular gene between JGI and DVI as per the Student’s ‘t’ test performed between the two groups. ‘*’- P<0.05; ‘**’- P<0.01; ‘***’-P<0.001.