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Distribution of Fusarium oxysporum f. sp. niveum and the expression of the defense-related genes in inoculated resistant and susceptible watermelon plants
Fusarium wilt of watermelon
Fusarium oxysporum f. sp. niveum
phenylalanine ammonia lyase
|摘要:||西瓜蔓割病係由Fusarium oxysporum f. sp. niveum (FON) 所引起，為西瓜栽培的主要限制因子之一。基於西瓜抗蔓割病之機制，於分子及組織學的層次尚未明瞭，故本研究利用對蔓割病分別具抗、感性之西瓜品系 (抗病之JSB與感病之Sugar Baby)，為材料，探討西瓜抗蔓割病之可能機制。將上述抗、感病西瓜種子播植於混有西瓜蔓割病菌之病土9天後 (day post inoculation, dpi)，利用PCNB選擇性培養基，分離植株根段與莖段內部的病原菌，藉此分析抗、感病植株內的帶菌情形。結果顯示，於88%的抗、感病植株根部都可分離到病原菌，然而只有54%的抗病植株莖部與66%之感病植株莖部可分離到病原菌；繼而以接種9天與35天後之抗、感病植株為材料，分別進行石蠟及徒手切片，以觀察病原菌在抗、感病植株中之分佈，並計算抗、感病植株於接種35天後，其木質部中病原菌的纏據率。根據組織切片的結果得知，在抗病植株之根部 (9及35 dpi) 與低部莖段 (近土表部份，lower stems；35 dpi) 中可見FON纏據於木質部；然而，在抗病植株之高部莖段 (近子葉部份，higher stems；35 dpi) 中，則沒有觀察到如感病植株中所見之病原菌纏據的現象。而於抗病植株之根段、低部及高部莖段中 (35 dpi)，病原菌纏據率則較感病植株為低。再者，持續種植在病土中的其他抗病植株於接種35天後仍然健康存活，但感病植株早已全數萎凋死亡。因此，將上述持續種植於病土中 (81天) 的抗病植株各部份組織，以PCNB選擇性培養基分離病原菌。結果顯示，僅可由根部和地基部中分離到病原菌，而莖部及子葉以上的組織皆無病原菌存在。故JSB品系抗西瓜蔓割病之機制可能在減少病原菌於其莖部內維管束中的纏據。除此之外，為了解酸性第三族幾丁質分解酵素 (WM-Chi) 及苯丙胺酸氨裂解酶 (PAL) 是否與抗蔓割病之機制相關，本研究以反轉錄聚合酵素連鎖反應分析其基因於抗、感病植株接種FON後的表現情形。結果顯示，不論是在抗病或感病植株中，PAL基因可經FON接種而誘導其表現，且在接種9天後之抗病植株中，其表現仍持續被誘導，然而，此時PAL基因的表現情形於感病植株中反而下降；另一方面，雖然WM-Chi基因之表現亦可受FON接種所誘導，但其於接種後之抗、感病植株中的表現情形並無差異。故推測PAL可能與西瓜抗蔓割病的機制有關，而WM-Chi則可能與之無關。|
Fusarium wilt of watermelon, caused by Fusarium oxysporum f. sp. niveum (FON), is one of the limiting factors on watermelon production. The molecular and histological mechanisms regarding to the Fusarium wilt resistance in watermelon, however, remain unclear so far. In this study, a Fusarium wilt resistant line (JSB), which was derived from mutation of susceptible Sugar Baby population in the watermelon fields, was used to investigate the resistance mechanism of watermelon to FON. The resistant and susceptible watermelon plants were grown in FON-infested soil and the pathogen was isolated from infected tissues at the 9th day post inoculation (dpi) by plating on PCNB selective media to compare the pathogen distribution patterns in resistant and susceptible plants. Results indicated that FON could be recovered from 88% of the roots in both watermelon lines, and from 54% and 66% of the stem segments in resistant and susceptible watermelon plants, respectively. Furthermore, plants inoculated for 9 and 35 days were also used for paraffin and free hand sectioning, respectively, to observe the pathogen distribution, and the ratio of pathogen colonization in xylem vessels at 35 dpi was calculated. According to the tissue sections, the xylems of roots (9 and 35 dpi) and lower stems (the portions close to soil surface, 35 dpi) in resistant watermelon plants were also colonized by FON as in the susceptible ones; nevertheless, no colonization within higher stems (the portions close to cotyledons, 35 dpi) was observed in the resistant plants. The ratios of FON colonization in xylem vessels of roots, lower and higher stems were much lower in resistant plants than in susceptible ones (35 dpi). In addition, even though the susceptible plants grown in infested soil were all dead by 35 dpi, the remaining resistant ones still survived thereafter. Therefore, the remaining resistant plants grown in infested soil (81 dpi) were also used to analyze pathogen distribution patterns by plating the tissues on PCNB selective media. Results showed that pathogens could be isolated from roots and shoot bases; whereas stems and tissue parts above cotyledons were free of pathogens. These phenomena suggest that the resistance in JSB may result in reducing FON colonization in the vascular systems of the host. Moreover, in order to understand whether the watermelon acidic class III chitinase (WM-Chi) or phenylalanine ammonia lyase (PAL) is related to the resistance mechanism against Fusarium wilt, the gene expression of these two proteins was analyzed using reverse transcription-polymerase chain reaction (RT-PCR). The expression of PAL was induced by FON in both resistant and susceptible plants at 3 and 6 dpi. Its expression maintained to 9 dpi in resistant plants but was down-regulated in susceptible ones. On the other hand, there was no difference in the expression level of WM-Chi in resistant and susceptible plants even though its expression was also induced by FON in both lines. The results revealed that PAL, but not WM-Chi, may involve in resistance of watermelon to Fusarium wilt pathogen.
|Appears in Collections:||植物病理學系|
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