Co-infection of wilt-resistant chickpeas by Fusarium oxysporum f. Matuo T; Sato K, On two new forms of Fusarium lateritum. Transactions of the Mycological Society of Japan, McKerral A,
|Published (Last):||16 November 2015|
|PDF File Size:||12.45 Mb|
|ePub File Size:||19.58 Mb|
|Price:||Free* [*Free Regsitration Required]|
All relevant data are within the paper and its Supporting Information files. Abstract 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 various 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-PCR , which 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, albeit its entry in the resistant plant, further proliferation is severely restricted providing an evidence of efficient defense mechanism in the resistant chickpea cultivar. Introduction 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 date, 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 and colonization patterns in wilt-susceptible and wilt-resistant chickpea cultivars. 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 ]. Root 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 treatments viz. The plants were lightly watered using autoclaved tap water every 2—3 days.
Evaluation of disease symptoms and tissue collection 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: a early stage 0 hpi to 7 dpi , b middle stage 7 to 14 dpi , and c late stage 14 to 28 dpi based on the morphological symptoms observed.
Two types of tissues were collected: whole roots and root fractions of approximately 2 inches in length, as well as two fractions of shoot till 2nd internode. Spore count was recorded using a haemocytometer [ 21 ]. Foc 2 transformation was performed according to Mullins et al [ 23 ] with some modifications [ 24 , 25 ].
An overnight grown single colony of A. Microconidia 1. These colonies were serially transferred five times in the selection medium to confirm stability of transformation, growth and morphology. Phenotypic characterization of wild type and transformed Foc 2 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 ].
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 wavelengths for excitation and emission, respectively.
Protein concentration was measured using Bradford assay with bovine serum albumin as standard [ 28 , 29 ]. 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 ].
Microscopic monitoring of pathogen progression in chickpea plants 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 plants 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. In planta pathogen quantification 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. Table 1 Primer sequences specific to Fusarium oxysporum f. Primer Name.
FUSARIUM OXYSPORUM F.SP.CICERI PDF
Description[ edit ] Fusarium oxysporum is a common soil inhabitant and produces three types of asexual spores : macroconidia, microconidia and chlamydospores. They are generally produced from phialides on conidiophores by basipetal division. They are important in secondary infection. They are formed from phialides in false heads by basipetal division. They are formed from hyphae or alternatively by the modification of hyphal cells. They are important as endurance organs in soils where they act as inocula in primary infection.
All relevant data are within the paper and its Supporting Information files. Abstract 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 various 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-PCR , which 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, albeit its entry in the resistant plant, further proliferation is severely restricted providing an evidence of efficient defense mechanism in the resistant chickpea cultivar.
Shakazil Strittmatter P The reaction sequence in electron transfer in the reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase system. Such hosts are known to utilize the altered redox state of the infected cell to transmit downstream defense signals . In the present study, enhanced expression of carbohydrate substrate transporters in incompatible interaction probably indicated its role in preventing fungal invasion. Fusarium verticillioides GBB1, a gene encoding heterotrimeric G protein beta subunit, is associated with fumonisin B-1 biosynthesis and hyphal development but not with fungal virulence. However at 28 dpi, the pathogen load significantly decreased in all the fractions. Peroxidase expression was increased by several-fold in WR plants compared to JG62 plants, with a sharp expressional drop at 3dpi. In JGI, pathogen load remained high in R1 till 24 hpi and dipped during 2 to 4 dpi.
PLoS One. Fusarium oxysporum f. Reactive oxygen species are known to play pivotal roles in pathogen perception, recognition and downstream defense signaling. But, how these redox alarms coordinate in planta into a defensive network is still intangible. Present study illustrates the role of Fusarium oxysporum f.