Evaluation of different protein extraction methods for banana (Musa spp.) root proteome analysis by two-dimensional electrophoresis.

Four protocols viz., the trichloroacetic acid-acetone (TCA), phenol-ammonium acetate (PAA), phenol/SDS-ammonium acetate (PSA) and trisbase-acetone (TBA) were evaluated with modifications for protein extraction from banana (Grand Naine) roots, considered as recalcitrant tissues for proteomic analysis. The two-dimensional electrophoresis (2-DE) separated proteins were compared based on protein yield, number of resolved proteins, sum of spot quantity, average spot intensity and proteins resolved in 4-7 pI range. The PAA protocol yielded more proteins (0.89 mg/g of tissues) and protein spots (584) in 2-DE gel than TCA and other protocols. Also, the PAA protocol was superior in terms of sum of total spot quantity and average spot intensity than TCA and other protocols, suggesting phenol as extractant and ammonium acetate as precipitant of proteins were the most suitable for banana rooteomics analysis by 2-DE. In addition, 1:3 ratios of root tissue to extraction buffer and overnight protein precipitation were most efficient to obtain maximum protein yield.

Phenylpropanoid enzymes, phenolic polymers and metabolites as chemical defenses to infection of Pratylenchus coffeae in roots of resistant and susceptible bananas (Musa spp.).

Activity differences of the first (phenylalanine ammonia lyase, PAL) and the last (cinnamyl alcohol dehydrogenase, CAD) enzymes of phenylpropanoid pathway in the roots of resistant (Yangambi Km5 and Anaikomban) and susceptible (Nendran and Robusta) banana cultivars caused by root lesion nematode, Pratylenchus coffeae, were investigated. Also, the accumulation of phenolics and deposition of lignin polymers in cell walls in relation to resistance of the banana cultivars to the nematode were analyzed. Compared to the susceptible cultivars, the resistant cultivars had constitutively significantly higher PAL activity and total soluble and cell wall-bound phenolics than in susceptible cultivars. The resistant cultivars responded strongly to the infection of the nematode by induction of several-time higher PAL and CAD enzymes activities, soluble and wall-bound phenolics and enrichment of lignin polymers in cell wall and these biochemical parameters reached maximum at 7th day postinoculation. In addition, profiles of phenolic acid metabolites in roots of Yangambi Km5 and Nendran were analyzed by HPLC to ascertain the underlying biochemical mechanism of bananas resistance to the nematode. Identification and quantification of soluble and cell wall-bound phenolic acids showed six metabolites and only quantitative, no qualitative, differences occurred between the resistant and susceptible cvs. and between constitutive and induced contents. A very prominent increase of p-coumaric, ferulic and sinapic acids, which are precursors of monolignols of lignin, in resistant cv. was found. These constitutive and induced biochemical alterations are definitely the chemical defenses of resistant cvs. to the nematode infection.

Genetic diversity of Fusarium oxysporum f.sp. cubense isolates (Foc) of India by inter simple sequence repeats (ISSR) analysis.

To find out the genetic diversity of Indian Foc isolates of banana, a total of 107 isolates of Fusarium which includes 98 Foc isolates obtained from different banana growing regions of India and seven Foc isolates belong to all known VCGs obtained from Australia and two non-pathogenic Fusarium oxysporum (npFo) isolates were subjected to ISSR analysis. In the initial screening of ISSR primers, out of 34, 10 primers which generated more polymorphic bands were selected for further analysis. The Phylogenetic analysis carried out based on the fingerprints obtained through ISSR analysis indicated the presence of wide genetic diversity among the Foc isolates of India and also its polyphyletic nature. Totally, seven different clusters were obtained and these clusters differentiated the Foc isolates of India based on the races/VCGs. Besides, the cluster analysis clearly distinguished the freshly emerged Foc strain obtained from cv. Grand Naine (Cavendish-AAA) and Poovan (Mysore-AAB) from the other Foc isolates. The non-pathogenic F. oxysporum isolates which have been included for comparison purpose also clustered separately. All these above said findings indicates for the first time the discriminatory power of ISSR to clearly distinguish and separate the Foc isolates according to its race/VCGs and also its virulence. This study would be useful not only to design and develop effective management strategies but also useful for quarantine purposes.

First Report on the Occurrence of a Virulent Strain of Fusarium Wilt Pathogen (Race-1) Infecting Cavendish (AAA) Group of Bananas in India.

Banana wilt disease caused by Fusarium oxysporum f. sp. cubense is one of the most significant threats to banana production worldwide. Strains of F. oxysporum f. sp. cubense have been grouped into race-1, -2, or -4 on the basis of differential virulence among different genotypes of banana. In India, though the disease is reported among susceptible varieties of races 1 and 2, the disease is not reported from Cavendish cultivars, which are the differential host to race-4. Recent surveys of the Cumbum areas (Theni District, Tamil Nadu) revealed symptoms (e.g., yellowing and drooping of leaves around the pseudostem and longitudinal splitting of pseudostem) on cv. Grand Naine (Cavendish group - AAA). F. oxysporum f. sp. cubense was recovered and single-spore isolates had characteristic white-to-purple aerial mycelia producing single-celled, oval microconidia in false heads on branched monophialides and sickle-shaped macroconidia with an attenuated apical cell and a foot-shaped basal cell. Pathogenicity was demonstrated on cv. Grand Naine by inoculation with sand maize meal inoculum (20 g per pot containing 106 spores per g). Vegetative compatibility, using 33 nit-M testers of all known vegetative compatibility groups (2), showed that nit-1 mutants generated from a wild strain of F. oxysporum f. sp. cubense isolated from cv. Grand Naine formed robust heterokaryons with nit-M tester 0124 of the Department of Employment, Economic Development and Innovation, Brisbane, Australia and also with nit-M tester obtained from an isolate of F. oxysporum f. sp. cubense from Karpuravalli (Pisang Awak-ABB). Further characterization of this new Cavendish strain was studied on the basis of volatile odor production (3) using VCGs 0125 for race-1 ('inodoratum group') and 0120 for race 4 ('odoratum group') as positive controls and sterile medium as a negative control. This new F. oxysporum f. sp. cubense strain of Cavendish belonged to 'inodoratum' group of F. oxysporum f. sp. cubense. Pathogenicity was demonstrated on potted plants (10 per cultivar) of cvs. Rasthali (Silk-AAB), Karpuravalli (Pisang Awak-ABB), Ney Poovan (AB), Poovan (Mysore-AAB), Red Banana (AAA), Nendran (French plantain-AAB), Monthan (ABB), and Grand Naine (Cavendish-AAA) by inoculation with sand maize meal inoculum (20 g per pot containing 106 spores per g) in three replicate experiments. Plants were uprooted 2 months postinoculation and disease severity was estimated by rating internal vascular discoloration in the corm (1). The result showed that all cultivars, except Red Banana and Nendran, had the highest rating for disease severity, 6. To our knowledge, this is the first report of a virulent strain of F. oxysporum f. sp. cubense VCG 0124 of race-1on Cavendish banana. References: (1) J. Carlier et al. Technical Guidelines Number 6. INIBAP, Montpellier, France, 2002. (2) J. C. Correll et al. Phytopathology 77:1640, 1987. (3) N. Y. Moore. Aust. J. Bot 39:161, 1991.