Pi-Fang Linda Chang

Development of the molecular methods for rapid detection and differentiation of Fusarium oxysporum and F. oxysporum f. sp. niveum in Taiwan

Fusarium wilt, caused by Fusarium oxysporum (Fo), is one of the most important fungal diseases worldwide. Like other plant pathogens, Fo displays specialized forms in association with its hosts. For example, F. oxysporum f. sp. niveum (Fon) is the damaging pathogen causing Fusarium wilt disease on watermelon, whereas F. oxysporum f. sp. cubense is the pathogen that infects banana. A rapid and reliable pathogen identification or disease diagnosis is essential for the integrated disease management practices in many crops. In this study, two new primer sets, Fon-1/Fon-2 and FnSc-1/FnSc-2, were developed to differentiate Fon and Fo, respectively. The PCR method using the novel primer sets has high sensitivity to detect Fon when the DNA concentration was as low as 0.01 pg or when the conidia number was as few as 5. In comparison with the published primer set, the Fon-1/Fon-2 primer set, derived from the sequence of OP-M12 random primer-amplified fragment, produced a 174 bp DNA fragment, and was more specific to Fon in Taiwan. In addition, with optimized PCR parameters, the molecular method using the Fon-1/Fon-2 primer set could directly detect Fon even when watermelon samples were collected in its early stage of disease development.

A molecular diagnosis method using real-time PCR for quantification and detection of Fusarium oxysporum f. sp. cubense race 4

The Fusarium genus causes devastating plant diseases worldwide, in which Fusarium oxysporum is the most serious crop pathogen. Disease monitoring is the basis of integrated pest management of any disease. The lack of rapid, accurate, and reliable device to detect and identify plant pathogens is one of the main limitations in integrated disease management. This study describes an efficient and quantifiable diagnosis method for the specific detection of F. oxysporum f. sp. cubense (Foc) race 4 in field-infected banana. With the optimized PCR parameters using the SCAR (sequence characterized amplified region) primers FocSc-1/FocSc-2 and a real-time PCR strategy, the developed method showed high reproducibility and was very sensitive to detect extremely low quantities of Foc genomic DNA (gDNA). We also found that Foc gDNA in severely symptomatic banana pseudostems and leaves were 6946-fold and 26.69-fold more than in those of mild-symptomatic banana, respectively.

Characterization of three Colletotrichum acutatum isolates from Capsicum spp.

Colletotrichum acutatum causes anthracnose on peppers (Capsicum spp.), resulting in severe yield losses in Taiwan. Fungal isolates Coll-153, Coll-365 and Coll-524 collected from diseased peppers were found to differ in pathogenicity. Pathogenicity assays on various index plants revealed that Coll-524 was highly virulent and Coll-153 was moderately virulent to three commercially available pepper cultivars. Both isolates induced anthracnose lesions and produced abundant conidia. Coll-365 was only weakly virulent on pepper fruit, where it caused small lesions and hardly produced conidia on pepper fruit. However, Coll-365 was highly pathogenic to tomato fruit and mango leaves, where it caused anthracnose lesions and formed acervuli and conidia. All three isolates showed similar abilities in the attachment and germination of conidia, formation of highly branched hyphae and appressoria, penetration of cuticles, and infection of epidermal cells on chili peppers. Coll-365 accumulated less turgor pressure in appressoria but produced higher levels of cutinase and protease activity than Coll-153 and Coll-524 did. All three isolates invaded the neighbouring cells through plasmodesmata in chili peppers and showed similar pectinase or cellulase activities in culture. However, the most virulent strain Coll-524 expressed stronger laccase activity and was more resistant to capsaicin compared to Coll-153 and Coll-365. The three isolates are different in numbers and sizes of double-stranded RNAs. Depending on the cultivar genotypes, cellular resistance of chili pepper to C. acutatum might rely on the ability to restrict penetration, colonization, or conidiation of the pathogen. We conclude that the differences in pathogenicity among the three C. acutatum isolates of pepper are attributed to their ability to colonize the host plant.

Histopathology comparison and phenylalanine ammonia lyase (PAL) gene expressions in Fusarium wilt infected watermelons

Fusarium wilt disease of watermelon (Citrullus lanatus (Thunb.) Matsum & Nakai), caused by Fusarium oxysporum f. sp. niveum (FON), is one of the limiting factors of worldwide watermelon production. In this study, a Fusarium wilt resistant watermelon JSB, which was derived from a spontaneous mutation of the susceptible Sugar Baby (SB), was used to investigate histopathology. The number and diameter of xylem vessels in the root (10 mm below the shoot base) of resistant JSB plants were significantly higher than those in susceptible SB plants. At 9 days post inoculation (dpi), using the plate assay on Nash-PCNB media, FON could be recovered from 86% of the roots in the symptomless plants of both watermelon lines, and from 55% and 64% of the stem segments (5 mm above the shoot base) in resistant and susceptible plants, respectively. In paraffin and free-hand tissue sections, at 8, 13, and 35 dpi, the xylem of roots and stems close to the soil surface in resistant watermelon JSB plants was also colonised by FON, but to a much lower percentage than the susceptible SB ones. No colonisation below the middle of stems was observed in the resistant JSB plants. The susceptible plants grown in infested soil were all dead by 35 dpi, while the resistant plants remained healthy. These observations suggest that reducing FON colonisation in the vascular systems of the host may contribute to the resistance in JSB. Furthermore, the higher expression of the phenylalanine ammonia lyase (PAL) gene in JSB induced by FON and the effects of PAL inhibitor on the resistance of JSB suggested that PAL is involved in resistance of watermelon to Fusarium wilt pathogen.

Genetic diversity and identification of race 3 of Fusarium oxysporum f. sp. lactucae in Taiwan

Fusarium wilt disease of lettuce (Lactuca sativa L.) is one of the major threats to the production of lettuce in Taiwan. This disease is caused by the vascular wilt pathogen Fusarium oxysporum f. sp. lactucae. It is important for the lettuce breeders to identify the pathological races of F. oxysporum f. sp. lactucae in the course of developing F. oxysporum f. sp. lactucae-resistant cultivars for growers. Recently, race 3 of F. oxysporum f. sp. lactucae was identified in Taiwan based on the results of pathogenicity tests against three differential lettuce cultivars, Patriot, Costa Rica No. 4, and Banchu Red Fire, along with some Japanese F. oxysporum f. sp. lactucae reference strains for comparison. In our collected F. oxysporum f. sp. lactucae isolates, most isolates from Taiwan were identified as race 1 except two isolates, Fola-10 and Fola-40 collected from Taoyuan County identified as race 3. This is the first report of F. oxysporum f. sp. lactucae race 3 outside of Japan. The PCR-based tagging technique with the four reference markers was used to confirm the results of pathogenicity tests. In addition, genetic diversity between races of F. oxysporum f. sp. lactucae was also observed according to the results of random amplification of polymorphic DNA (RAPD) fingerprintings.

Cell wall reinforcement in watermelon shoot base related to its resistance to Fusarium wilt caused by Fusarium oxysporum f. sp. niveum

Fusarium wilt of watermelon, caused by Fusarium oxysporum f. sp. niveum, is one of the limiting factors for watermelon production in Taiwan. In recent research, the phenylalanine ammonia lyase (PAL) gene expressed in the shoot base of the Fusarium wilt resistant line JSB was related to Fusarium wilt resistance. Phenylalanine ammonia lyase is the key regulatory enzyme in the phenylpropanoid metabolic pathway. The downstream products of phenolic compounds are considered to be involved in the complicated plant defence mechanisms. They could act as signal molecules, antimicrobial substances and/or structural barriers. To study the resistant mechanisms of Fusarium wilt, the resistant JSB line was examined for comparison of F. oxysporum-watermelon interactions with the susceptible Grand Baby (GB) cultivar. Unlike infected GB, which was seriously colonized by F. oxysporum in the whole plant, the pathogen was limited below the shoot base of inoculated JSB, suggesting that the shoot base of JSB may contribute to Fusarium resistance. The data indicated that a significant increase in PAL activity was found in shoot bases of the resistant JSB line at 3, 9, 12 and 15 days after inoculation (DAI). Shoot bases of resistant watermelons accumulated higher amounts of soluble and cell wall-bound phenolics at 3–9 DAI; the susceptible GB cultivar, however, only increased the cell wall-bound phenolics in shoot bases at 3 DAI. High lignin deposition in the cell walls of vascular bundles was observed in the shoot bases of JSB but not of GB seedlings at 6 and 9 DAI. In the roots and shoot bases of JSB seedlings at 6 DAI, peroxidase enzyme activity increased significantly. In summary, the results suggest that accumulation of cell wall-bound phenolics and increase of peroxidase activity in shoot bases of JSB seedlings during F. oxysporum inoculation, together with the rapid deposition of lignin in the cell walls of vascular bundles, may have provided structural barriers in resistant JSB line to defend against F. oxysporum invasion.