Present study illustrates the role of Fusarium oxysporum ciceri Race1 (Foc1) induced redox responsive transcripts in regulating. Abstract. Based on the differential reaction of 10 chickpea cultivars to pathogenic isolates of Fusarium oxysporum f. sp. ciceri, the existence of at. About ha are sown annually to chickpea (Cicer arietinum L.) in Andalucia, southern Spain, approximately 66% of the total national acreage of the crop.

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Conceived and designed the experiments: Fusarium wilt caused by Fusarium oxysporum f. Understanding the molecular basis of chickpea-Foc interaction is necessary to improve chickpea resistance to Foc and thereby the productivity of chickpea. We transformed Foc race 2 using green fluorescent protein GFP gene and used it to characterize pathogen progression and colonization in wilt-susceptible JG62 and wilt-resistant Digvijay chickpea cultivars using confocal microscopy.

We also employed quantitative PCR qPCR to estimate the pathogen load and progression across fusariim tissues of both the chickpea cultivars during the course of the disease. Additionally, the expression of several candidate pathogen virulence genes was analyzed using quantitative reverse transcriptase PCR qRT-PCRwhich showed their characteristic expression in wilt-susceptible and resistant chickpea cultivars.

Our results suggest that the pathogen colonizes the susceptible cultivar defeating its defense; however, f.sp.cicer its entry in the resistant plant, further proliferation is severely restricted providing an evidence of efficient defense mechanism in the resistant chickpea cultivar. Chickpea Cicer arietinum L. Chickpea yield has been mostly stagnant over the years due to its susceptibility to various biotic and abiotic factors.

The important biotic factors affecting chickpea productivity include Fusarium wilt caused by Fusarium oxysporum f. Eight races have been reported for Fusarium oxysporum f. The pathogen can survive in soils for up to six years even without the host, which makes its control very difficult [ 5 ]. Conventional strategies, such as crop rotations, avoiding the infected field to grow chickpea and the use of chemical fungicides are being used to manage the disease. However, they have not been successful in controlling the disease [ 6 ].

Plant-pathogen interaction is complex and involves the expression of both, pathogen virulence genes as well as plant defense genes. Till oxyxporum, various candidate genes with prime role in fungal pathogenesis have been identified [ 7 ]. These fungal pathogenicity genes are categorized based on formation of infection structures, cell wall degradation, toxin biosynthesis, signaling and proteins suppressing plant defense [ 8 — 10 ]. Signaling genes expressed during pathogenesis such as fmk1 a mitogen-activated protein kinase in F.

In the present study, we transformed Foc race 2 with the eGFP gene encoding green florescent protein GFP and used it to understand the infection process f.sp.cieri colonization patterns in wilt-susceptible and wilt-resistant chickpea cultivars.

Plant Disease | Races of Fusarium oxysporum f. sp. ciceri

The expression of several pathogen virulence related genes involved in processes like signaling, cell wall degradation and fungal morphogenesis, as well as those identified in previous studies to be important for fungal pathogenesis was also analyzed. All the three approaches revealed similar and significant differences among the wilt-susceptible and wilt-resistant chickpea cultivars.

JG62 selection from germplasm is susceptible to Fusarium wilt, while Digvijay Phule G X Bheema is resistant to the disease [ 7 ]. Fuzarium tips of tap root and lateral roots were cut and the entire root system was dipped in spore suspension for 5 min. Plants mock-inoculated with sterile deionized water served as controls. Thus four oxyporum viz. The plants were lightly watered using autoclaved tap water every 2—3 days.

The plants were evaluated for morphological changes and development of wilting symptoms daily after inoculation. Tissues of all the four treatments were collected at all the eight time points mentioned above. The time scale of the infection process was divided as: Two types of tissues were collected: A kill curve was initially set up for Foc using the poisoned food technique [ 20 ] to determine the minimum inhibitory concentration of hygromycin B.


Spore count was recorded using a haemocytometer [ 21 ]. Foc 2 transformation was performed according to Mullins et al [ 23 ] with some modifications [ 2425 ]. An overnight grown single colony of A. These colonies rsces serially transferred five times in the selection medium to confirm stability of transformation, growth and morphology.

The Foc 2 transformants were transferred to PDA containing hygromycin B to observe colony morphology and cultural characteristics with respect to wild-type. Slide preparations using a drop of sterile water and hyphae were done for both the wild-type and the transformants.

The radial mycelial growth RMG was determined by measuring the length of four radii each radius in one direction daily. The radial growth rate RGR was calculated by the slope of the linear regression of the mean colony radius over time [ 27 ].

Races of Fusarium oxysporum f.sp. ciceri in Andalucia, southern Spain [1985]

In addition, pathogenicity of the transformants was evaluated in comparison to the wild type. Microconidial suspensions of wild-type Foc 2 and five transformants were used to inoculate the susceptible JG62 and resistant Digvijay chickpea cultivars as described earlier. Three replicates of five seedlings per cultivar per transformant as well as the wild-type were inoculated. The seedlings mock inoculated with sterile deionized water served as control.

The cultures were centrifuged at rpm and the pelleted mycelia were crushed under liquid nitrogen and transferred to 5 ml extraction buffer 10 mM Tris pH 7.

Mycelial debris was removed by another round of centrifugation. The resulting supernatant was assayed for fluorescence using nm and nm ffusarium for excitation and emission, respectively. Protein concentration was measured using Bradford assay with bovine serum albumin as standard [ 2829 ].

Relative fluorescence units RFU values were then normalized with respect to protein concentration. Mycelial mass collected by filtration through muslin cloth was crushed to fine powder under liquid nitrogen and DNA was isolated using modified CTAB protocol [ 30 ].

Another set of JG62 and Digvijay plants was inoculated with the selected transformant D4. The inoculated and control chickpea plants were sampled daily during 1 to 4 DPI and at a 2—3 day interval thereafter, up to 18 DPI. During each sampling, four rsces were collected from each treatment.

The entire surface of the tap and lateral roots of each plant was observed under a confocal laser scanning microscope CLSM. In addition, auto fluorescence of chickpea plants was assessed at wavelengths of — nm. Three sets of primers viz.

PCR conditions were optimized for each primer pair and all the reactions were performed at least twice. The amplification products were electrophoresed and visualized using a gel documentation system Syngene, USA. To determine pathogen load in susceptible JG62 and resistant Digvijay chickpea plants, genomic DNA isolated from whole roots of inoculated plants was used as template for qPCR. Non-template control as well as DNA from un-inoculated chickpea root served as negative controls.

Each reaction contained 30 ng DNA, 0. The exponential phase of the reaction was identified by plotting the fluorescence on a log scale and linear regression analysis was performed to estimate the efficiency of each reaction using Linreg software [ 32 ]. The amount of pathogen DNA was estimated in the roots of inoculated cultivars at different time points from the standard curves established earlier.

This revealed the biomass of the pathogen at respective time-points. Similar procedure was followed for estimating the biomass of the pathogen in the fractions of chickpea root and shoot tissues 2 inch fractions from the root tip clipped for inoculation at different time points. The lowest 2 inch fraction of the root was named as R1, while the topmost 2 inch fraction was named as R5.

Similarly, S1 was the lowest shoot fraction, followed by S2. JG62 has very short root-length and root mass compared to Digvijay. For candidate gene expression analysis, gene specific primers were designed Table 2 from the conserved regions of fungal virulence related genes using the sequences available in NCBI database database-fungi http: Wilting symptoms started to appear at about 7 dpi in JGI and intensified with time.


The presence of hygromycin B phophotransferase hph and eGFP genes in the transformants was confirmed by PCR amplicons of sizes bp and 1 kb for hph and bp for eGFPrespectively; while no amplification was observed in wild-type Foc 2 S5 Fig. No morphological changes in size or shape of vegetative structures were observed. The transformants retained the colony morphology characteristics of the wild-type including white cottony growth of aerial mycelia. Similarly, virulence of all the five transformants was comparable to that of the wild type.

The level of GFP fluorescence in the five transformants was variable, while the wild-type Foc 2 showed negligible fluorescence. Radial growth rate; calculated by slope of linear regression of the mean colony radius over time.

Each value is the mean of three replicates petri dishes.

Races of Fusarium oxysporum f. sp. ciceri – [email protected]

In early stage of infection, colonization on root surface was observed in both the cultivars by forming the primary mycelia at the root apex Fig 1B—1D. Surface colonization was followed by direct penetration of hyphae into epidermal cells without forming any specialized structures Fig 1B Inset. The pathogen then entered root cortex region by 2 dpi Fig 1E.

Uniform expression of eGFP in hyphae and spores of transformed isolate D4. Germinating conidium with primary mycelium in contact with root apex at 24 hpi. Initial hyphal colonization at lower root zone at 2 dpi. Intermediate root zone showing hyphal colonization extending from epidermis to cortical cells at 2 dpi. Vascular region of root getting colonized at 3 dpi.

Fungal colonization in cortex region of DVI. Based on these pilot studies, in-depth analysis of pathogen infection in JGI and DVI was performed throughout the disease progression.

During early infection stages up to 4—6 dpiboth cultivars showed surface colonization and entry of the pathogen in lower roots Fig 2A and 2B. However, substantial colonization of vascular region was thereafter observed in lower and middle root zone of only JGI at 8 dpi Fig 2C. Further, the appearance of wilting symptoms in JGI was marked with heavy colonization of lower, middle and upper root zones along with the lower stem region at 10—12 dpi Fig 2D.

However in DVI, initially the pathogen was restricted to root cortex region Fig 2C and 2D and reached xylem vessels very late by 18 dpi and that too in very less numbers. By 25 dpi, both the root and stem of JGI were heavily colonized resulting in disruption of normal architecture, complete wilting and death of most of the plants; while the root and shoot architectures of DVI plants were nearly normal Fig 3B and 3C.

Heavy colonization of root of susceptible JG62 plant in longitudinal section. 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. Longitudinal section of stem of susceptible JG62 plant with the extensive presence of conidia as well as mycelia of fungus.

Cross and longitudinal sections of root of resistant plant Digvijay with absence of any fungus and normal architecture of roots. Longitudinal and cross section of stem of resistant Digvijay plant without any fungal infection. At this time point, the maximum amount of pathogen DNA was detected in all the four R1-R4 root fractions and both S1-S2 the shoot fractions. However at 28 dpi, the pathogen load significantly decreased in all the fractions. Interestingly, the pathogen load was below the detectable limit in R3 and Fusariu, fractions at 28 dpi oxyspotum DVI.