摘要
西瓜枯萎病是导致西瓜连作障碍的最主要因素之一,严重制约着西瓜生产的发展。为得到西瓜枯萎病生物防治高效菌株,对西瓜枯萎病生防放线菌进行了筛选,然后探讨了生防放线菌对西瓜的防病促生作用。主要得出以下结果:
1.利用柯赫氏法则对靶标病原菌进行了验证,确定X1、X2、X3均为尖孢镰刀菌西瓜专化型。经过致病性检验,确定X1致病性最强且发病潜伏期最短,可以作为后续实验的致病菌和生物活性测定的靶标菌。
2.利用琼脂块法对本研究保藏的100余株拮抗性放线菌进行了皿内复筛,获得21株拮抗性较强的菌株,其中菌株Act1和28G336B对X1的抑菌圈直径分别达到24 mm和19 mm;采用生长速率法测定了其中14株放线菌无菌发酵滤液对供试靶标菌的相对抑菌率,结果表明:发酵液对靶标菌均有抑制作用,最高抑制率可达85.7 %。
3.供试放线菌孢子悬液对西瓜幼苗有一定的防病作用。Act1、Act11和Act12对西瓜植株均无致病性,且Act1处理可以抑制X1对西瓜植株的侵染,Act11和Act12处理在一定程度上延缓了X1对西瓜植株的侵染。
4.供试放线菌孢子悬液和活菌制剂对西瓜生长有一定的促进作用。用Act1、Act11和Act12孢子悬液接种时,西瓜株高和干重较对照的最大增幅分别为32.8 %和66.7 %;用活菌制剂接种时,西瓜根系鲜重和干重的最大增幅分别为59.3 %和59.8 %。
5.接种放线菌活菌制剂对西瓜植株保护性酶PPO活性和可溶性蛋白质含量有显著影响。接种可使西瓜叶片和根系PPO活性分别提高2.0 %~81.8 %和0.6 %~13.6 %;用相对抑菌率﹥60 %的5株放线菌发酵液进行叶面喷施,可使西瓜叶片PPO活性提高8.7 %~30.6 %。PPO活性提高有利于提高西瓜的抗病性。接种活菌制剂导致西瓜叶片和根系可溶性蛋白质含量下降,降幅分别为9.2 %~52.5 %和36.7 %~83.3 %。
6.连作西瓜土传病害的发生与根域微生物生态失调有关。连作罹病与新茬健康西瓜根域微生物数量及根域真菌区系组成差异明显。连作2年西瓜病株根域细菌、真菌及放线菌总量分别较健株增加128.0 %、55.1 %及90.8 %,根表真菌数量较健株增加126.0 %;病株根表和根区的镰刀菌数量约分别为健株的4倍和5倍。健康西瓜植株根域真菌种类呈多样性,而罹病西瓜植株根域真菌区系中镰刀菌占绝对优势,其他真菌数量大幅度减少,根域微生物区系已转化为病理组合,增大了西瓜根部土传病害发生的机率。
7.接种生防放线菌对西瓜根域微生物区系有显著影响。Act1接入西瓜育苗基质,西瓜根域细菌数量较对照提高48.7 %~651.6 %,其中西瓜根表土壤中芽孢杆菌数量高达对照的8倍;放线菌数量提高17.8 %~478.4 %;真菌数量减少29.2 %~45.2 %,其中西瓜根区和根表土壤中镰刀菌密度较对照分别降低55 %和60 %。B/F较对照提高75.4 %~369.5 %。Act1具有较好的定殖稳定性,接种100 d后,在西瓜根域土壤中活菌数仍高达106·g-1以上。接种生防菌促使西瓜根域土壤由真菌型向细菌型转变,在西瓜根域形成含有大量拮抗性放线菌、可抗御西瓜枯萎菌等土传病害的生物屏障。
Watermelon fusarium wilt is one of the most important factors of the continuous cropping obstacle of watermelon, which is seriously restricting the development of watermelon production. In order to obtain some high efficient actinomyces strains against watermelon fusarium wilt, some biocontrol actinomyces were screened. Then their prophylaxis and growth promoting effect on watermelon was studied.The result showed as following:
1. In this paper, target pathogens were verified according to Koch’s postulate. The results showed that both X1、X2、X3 were Fusarium oxysporum schlechlendahl f. sp. melons (Leach at currence) snyder et Hansen. Pathogenicity test showed that pathogenic intensity X1>X3>X2, the pathogenesis latent period of X1 was shortest, so it can be used as pathogens and target pathogens of biological activity assay for the further experiments.
2. During the screening of more than 100 strains antagonistic actinomycetes by agar block method on culture dishes, 21 strains strongly antagonistic actinomycetes were obtained. The antibacterial circle diameter of Act1 and 28G336B respectively reached 24 mm、19 mm. The inhibition rate of fermentation broth against target pathogens of 14 actinomyces strains were determined by plate growth rate method. The results showed that all of fermentation broth had inhibition, the highest being 85.7 %.
3. All of the inoculation with spore suspension of actinomyces had prophylaxis effect on watermelon seedling. Act1, Act11 and Act12 had no pathogenicity for watermelon. The infection of X1 could be inhibited by treatment of Act1 and delayed by treatment of Act11 and Act12.
4. Both inoculation with spore suspension and live actinomyces preparation had promoting effect on watermelon.The plant height and dry weight significantly increased 32.8 % and 66.7 % in the treatment of inoculation with spore suspension of Act1、Act11 and Act12 compared with CK.The plant height and dry weight significantly increased 59.3 % and 59.8 % in the treatment of live actinomyces preparation compared with CK.
5. Inoculation with live actinomyces preparation had significant effect on the PPO activity and the soluble protein content of watermelon.The PPO activity of leaf and root respectively increased 2.0 %~81.8 % and 0.6 %~13.6 % in treatment of live actinomyces preparation.The PPO activity of leaf increased 8.7 %~30.6 % in treatment of foliage spray by fermentation broth of 5 actinomyces strains.The promotion of PPO activity was beneficial to improving disease resistance of watermelon. Inoculation with live actinomyces preparation lead to the desease of the soluble protein content of watermelon. The soluble protein content of leaf and root respectively decreased 9.2 %~52.5 % and 36.7 %~83.3 % in treatment of live actinomyces preparation.
6. The dysbiosis of watermelon rooting zone leaded to soil borne disease.There were significant differences of microbe quantity and fungi flora between alternate cropping healthy and continuous cropping diseased watermelon rooting zone. The results showed that, the quantity of bacteria、fungi and actinomyces of rooting zone of watermelon continuous cropping for 2 years were rifely higher than those in the alternate cropping healthy CK. The quantity of bacteria、fungi、fungi of root surface、actinomyces increased 128.0 %、、55.1 %、126.0 %、90.8 % respectively. The fusarium number of rhizosphere soil and root surface soil of continuous cropping diseased watermelon plant was about 5 and 4 times higher than that of alternate cropping healthy watermelon plant. The fungi species of healthy watermelon rooting zone showed more multiplicity. The physiological combination of rooting zone micro-community keeps normal. Fusarium was a dominant species in fungi flora of diseased watermelon rooting zone, while other fungus quantity reduced quickly. The rooting zone micro-community of diseased watermelon had transformed into pathological combination, which increase the rate of soil borne disease of watermelon root.
7. Inoculating with bio-control actinomyces on microbial flora of watermelon rooting zone had significant effect. The results showed that inoculating with Act1 in nursery bed soil had raised bacteria and actinomyces number of rooting zone soil of watermelon, reduced fungi number of rooting zone soil of watermelon. The quantity of bacteria increased by 48.7 %~651.6 %, thereinto the quantity of gemma bacillus of watermelon root surface soil was 8 times more than CK, the quantity of actinomyces increased by 17.8 %~478.4 %, the quantity of fungi decreased by 29.2 %~45.2 %, thereinto the destiny of Fusarium in the watermelon rhizosphere soil and root surface soil decreased markedly by 55 %and60 %, respectively. B/F increased by 75.4 %~369.5 %. 100 days after inoculation, the number of Act1 was more than 106·g-1. These results demonstrate an expectant colonization stability of Act1. After inoculation with Act1, the microbial flora of watermelon rooting zone soil changed markedly, it changed from fungi type to bacteria type. In watermelon rooting zone soil, a biological barrier formed mostly by bio-control actinomyces against soil borne plant diseases such as watermelon Fusarium wilt.
引文
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