Antifungal effects of iron sulfate on grapevine fungal pathogens

The present study aimed to determine the most efficient experimental conditions of iron sulfate use leading to optimal inhibition in the development of fungal pathogens. Assays have been focused on fungal species inducing severe grapevine diseases. FeSO₄ directly inhibited the in vitro mycelial growth of Botrytis cinerea, Eutypa lata, Phaeomoniella chlamydospora, Phaeoacremonium aleophilum, Diplodia seriata, and Neofusicoccum parvum with variable efficiency in the range of 0.5–10 mM. The development was always completely inhibited at 20 mM. This inhibitory effect was greatly increased at acidic pH values. The anionic moiety of the molecule was of importance since bromide, chloride and sulfate were highly active, whereas acetate and oxalate showed a small effect. Electron microscope observations on E. lata and B. cinerea showed that a treatment with FeSO₄ induced dramatic changes in the hyphal organization leading to cell death. No toxicity was observed on grapevine leaves following repeated FeSO₄ sprays in the antifungal concentration range. Therefore, FeSO₄ may be proposed to effectively replace the long-term pollutant use of CuSO₄ as an antifungal agent, with the additional advantage of iron being an important plant micronutrient.     

灰葡萄孢菌胞外大分子分泌物的植物毒性及漆酶性质鉴定

采用透析的方法提取灰葡萄孢菌分泌的大于14 kD的胞外大分子物质,将其接种到番茄和菊花叶片组织,检测灰葡萄孢菌胞外大分子分泌物的植物毒性,并以鞣酸和RB亮蓝为指示剂鉴定灰葡萄孢菌胞外大分子分泌物的漆酶性质。结果显示:灰葡萄孢菌胞外大分子分泌物可导致叶片组织坏死,具有植物毒性;接种灰葡萄孢菌后第3天,鞣酸和RB亮蓝平板出现不同程度的显色反应,第6天时平板几乎完全显色,胞外大分子物质具有漆酶性质。     

Long‐lasting β‐aminobutyric acid‐induced resistance protects tomato fruit against Botrytis cinerea

Minimizing losses to pests and diseases is essential for producing sufficient food to feed the world's rapidly growing population. The necrotrophic fungus Botrytis cinerea triggers devastating pre‐ and post‐harvest yield losses in tomato (Solanum lycopersicum). Current control methods are based on the pre‐harvest use of fungicides, which are limited by strict legislation. This investigation tested whether induction of resistance by β‐aminobutyric acid (BABA) at different developmental stages provides an alternative strategy to protect post‐harvest tomato fruit against B. cinerea. Soil‐drenching plants with BABA once fruit had already formed had no impact on tomato susceptibility to B. cinerea. However, BABA application to seedlings significantly reduced post‐harvest infection of fruit. This resistance response was not associated with a yield reduction; however, there was a delay in fruit ripening. Untargeted metabolomics revealed differences between fruit from water‐ and BABA‐treated plants, demonstrating that BABA triggered a defence‐associated metabolomics profile that was long lasting. Targeted analysis of defence hormones suggested a role of abscisic acid (ABA) in the resistance phenotype. Post‐harvest application of ABA to the fruit of water‐treated plants induced susceptibility to B. cinerea. This phenotype was absent from the ABA‐exposed fruit of BABA‐treated plants, suggesting a complex role of ABA in BABA‐induced resistance. A final targeted metabolomic analysis detected trace residues of BABA accumulated in the red fruit. Overall, it was demonstrated that BABA induces post‐harvest resistance in tomato fruit against B. cinerea with no penalties in yield.     

2-噻唑酰氨基环己烷基磺酰胺的合成与杀菌活性

为进一步研究环烷基磺酰胺类化合物的杀菌活性与构效关系,在前期工作基础上,合成了11个未见文献报道的2-噻唑酰氨基环己烷基磺酰胺类化合物 ( 7a ~ 7k ),其结构均经1H NMR、13C NMR、质谱和元素分析确证。分别采用菌丝生长速率法、黄瓜活体叶片法、孢子萌发法和番茄活体盆栽法对目标化合物进行了生物活性测定。结果表明:目标化合物对番茄灰霉病菌Botrytis cinerea表现出较好的抑制活性,其中化合物 7a 和 7c 在10 mg/L下对番茄灰霉病菌孢子萌发的抑制率分别为90%和67%;在200 mg/L 施药剂量下,对活体黄瓜叶片、番茄叶片和番茄花上灰霉病的防治效果,化合物 7a 分别为75%、78%和30%,化合物 7c 分别为78%、62%和44%,均优于对照药剂腐霉利,有进一步研究的价值。     

海洋生境贝莱斯芽孢杆菌TCS001的鉴定及抑真菌活性

为研究海洋生境芽孢杆菌TCS001的分类地位和抑菌活性,通过形态和生理生化特征观察,并结合gyrA序列同源性分析对菌株进行了鉴定;通过平板对峙培养法测定了菌株TCS001对多种植物病原真菌的抑菌谱;采用菌丝生长速率法和凹玻片法,测定了不同浓度TCS001菌株发酵滤液对靶标菌黄瓜灰霉病菌Botrytis cinerea菌丝生长和孢子萌发的影响。结果显示:该菌株为贝莱斯芽孢杆菌Bacillus velezensis,其对6种供试病原菌均有一定的抑制效果,其中对黄瓜灰霉病菌的抑制率最高,达87.66%。不同稀释倍数下,TCS001发酵滤液对黄瓜灰霉病菌均有一定的抑制作用,其中,稀释5倍时对菌丝生长和孢子萌发的抑制率最高,分别为96.24%和98.05%,稀释20倍时抑制率也均达90%以上。形态学观察发现,TCS001发酵滤液可导致黄瓜灰霉病菌孢子萌发芽管中间或顶端膨大畸形。研究表明,海洋生境贝莱斯芽孢杆菌TCS001极具开发为微生物农药的潜能。     

灰葡萄孢产孢相关基因BC1G_03293功能的初步研究

据灰葡萄孢菌丝发育和菌核形成时期的RNA-seq数据,挑选出在这2个时期表达量差异较大的基因BC1G__03293进行基因功能的研究。结果发现,BC1G__03293在菌核发育时期表达明显上调,表达量比菌丝生长时期上调了70倍以上。该基因编码的蛋白质含有229个氨基酸,N端包含信号肽,但不含有任何已知保守结构域。通过同源重组的方法得到了BC1G__03293的敲除转化子ΔBC1G__03293-2和ΔBC1G__03293-4,并运用农杆菌转化技术得到了互补转化子ΔBC1G__03293-2-C2和ΔBC1G__03293-2-C3。BC1G__03293基因敲除后生长、致病及菌核形成等表型无明显变化,但敲除转化子的分生孢子产量显著下降,仅为野生型菌株B05.10分生孢子产量的45%,并且BC1G__03293基因互补可使敲除转化子的产孢量得到明显恢复。结果说明BC1G__03293基因与灰葡萄孢的分生孢子形成相关。     

Triggering the biocontrol of Botrytis cinerea by Trichoderma harzianum through inhibition of pathogenicity and virulence related proteins

This study reports a strain of Trichoderma harzianum CCTCC-SBW0162 with potential to enhance biocontrol activity against gray mold pathogen, Botrytis cinerea, and with a pivotal role in tomato (Solanum esculentum) plant growth enhancement. A total of 254 Trichoderma isolates were screened by in vitro antagonistic assay. Of these, 10 were selected for greenhouse experiments based on their greater inhibition of B. cinerea. The in vitro antagonistic assay and greenhouse experiments indicated that T. harzianum CCTCC-SBW0162 gave the highest inhibition rate (90.6%) and disease reduction (80.7%). Also, to study the possible mechanism associated with antifungal activity of CCTCC-SBW0162 against B. cinerea, molecular docking was used to assess the interactions between CCTCC-SBW0162-derived metabolites, and pathogencity and virulence related proteins of B. cinerea. The molecular docking results indicated that the combination of harzianopyridone, harzianolide and anthraquinone C derived from CCTCC-SBW0162 could synergistically improve antifungal activity against B. cinerea through the inhibition/modification of pathogenicity and virulence related proteins. However, this computerized modeling work emphasized the need for further study in the laboratory to confirm the effect T. harzianum-derived metabolites against the proteins of B. cinerea and their interactions.     

Fusaricidins in Paenibacillus polymyxa A21 and their antagonistic activity against Botrytis cinerea on tomato

Four kinds of antifungal compounds from an extract of Paenibacillus polymyxa A21 with molecular masses of 883.56, 897.59, 947.55, and 961.58 Da were characterized as the members of fusaricidin-type of antibiotics according to LC-MS analysis. Fusaricidins isolated from culture filtrate displayed high antagonistic activity against several plant fungal pathogens, especially Botrytis cinerea, the causal agent of gray mold. The fusaricidins biosynthetic gene cluster (BGC) from A21 was cloned by PCR and comparative cluster analysis revealed that gene fusTE, the 3′ boundary of the fusaricidin BGC in strain PKB1, was not present in fusaricidin BGC of A21, indicating that fusTE is not necessary for fusaricidin synthesis. Fusaricidin extract from A21 significantly reduced gray mold disease incidence and severity on tomato. The mRNA levels for three patho-genesis-related proteins (PRs) revealed that treatment of tomato leaves with fusaricidin extract induced the expression of PR genes to different levels, suggesting that one reason for the reduction of gray mold infection by fusaricidin is induction of PR proteins, which lead to increased resistance to pathogens. This is the first report of the application of fusaricidins to control tomato gray mold and the comparative cluster analysis provides important molecular basis for research on fusaricidin biosynthesis.     

北京地区草莓灰霉病菌对异菌脲的抗性及抗性分子机制

由灰葡萄孢(Botrytis cinerea Pers.)引起的草莓灰霉病是草莓上危害严重的病害之一。为了解北京地区草莓灰霉病菌对二甲酰亚胺类常用杀菌剂异菌脲的抗性,本研究采用最低抑制浓度法分别检测了2013年和2014年从北京地区15个草莓园采集的共计121株草莓灰霉病菌对异菌脲的抗性。结果表明,北京地区草莓灰霉病菌对异菌脲存在较高的抗性频率,2014年较2013年的抗性频率略有上升,由40.4%上升为45.3%。不同草莓园菌株的抗性频率差异很大,可能与用药水平有关。2014年的低抗、中抗和高抗菌株数分别占检测菌株总数的9.4%、28.1%和7.8%。利用PCR技术扩增编码组氨酸激酶基因BcOS1中与二甲酰亚胺抗性相关的区段,对抗性菌株的分子机制进行了初步研究,结果表明BcOS1基因第1 214位核苷酸发生了2类突变:以第Ⅰ类为主,菌株的抗性水平为中抗和高抗,由ATC突变为AGC,导致第365位氨基酸由异亮氨酸变为丝氨酸;第Ⅱ类菌株为低抗,由ATC突变AAC,导致第365位氨基酸由异亮氨酸变为天冬酰胺。