<i>Bacillus subtilis Strains</i>with Antifungal Activity against the Phytopathogenic Fungi

Bacillus strains isolated from the rhizosphere soil of potato roots were evaluated for the potential antagonistic activity against fungal pathogens in vitro and in vivo. Two bacterial isolates were identified as new Bacillus subtilis strains by 16S rRNA and GyrB gene sequencing and were designated GM2 and GM5, respectively. Strains were characterized by their ability to inhibit growth of a number of phytopathogenic fungi. It was shown that GM5 strain inhibited growth of phytopathogenic fungi more effectively than GM2 strain. Both strains were capable of producing a number of hydrolytic enzymes as well as antimicrobial metabolites (ammonia and HCN). In addition, GM2 strain also produced siderophores. Four genes encoding antimicrobial peptides were identified in the genome of GM2 strain: ituC, bmyB, bacA and srfA. Genome of GM5 contained two genes encoding for antimicrobial peptides, srfA and fenD. Purified lipopeptide fraction from GM5 but not from GM2 strain was able to control Fusarium solani spread in the plate assay. Furthermore, Bacillus subtilis strain GM2 promoted growth of wheat but only GM5 strain was able to protect wheat seedlings from Fusarium oxysporum infection.

PCR-Mediated Detection of Endophytic and Phytopathogenic Fungi from Needles of the Japanese Black Pine, <i>Pinus thunbergii</i>

A specific and sensitive polymerase chain reaction (PCR) assay based on the internal transcribed spacer (ITS) region of rDNA sequences was developed to detect endophytic and phytopathogenic fungi from needles of the Japanese black pine, Pinus thunbergii. Sequences of the ITS regions of Lophodermium conigenum, Lecanosticta acicola, Pestalotiopsis neglecta, Rhizosphaera kalkhoffii, and Septorioides pini-thunbergii were compared, and each specific primer pair for these species was designed. First, the designed primer pairs were tested for their specificity to detect each species. A PCR product was amplified only each combination of species and its specific primer pair, confirming the specificity of the designed primer pairs. These primer pairs were also tested on DNA extracted from the needles of P. thunbergii. The PCR products were amplified not only in needles with lesions but also in healthy needles without symptoms. Furthermore, several endophytic and phytopathogenic fungi could be simultaneously detected from the same region in a needle. The PCR-mediated detection method developed in this study will be a valuable tool for the detection of the endophytic and phytopathogenic fungi, not only as a rapid diagnostic tool for early detection but also for monitoring variations in both the quality and quantity of the endophytic and phytopathogenic fungi in needles in Japanese black pines.

Isolation and Screening of Silicate Bacteria from Various Habitats for Biological Control of Phytopathogenic Fungi

Silicate solubilizing bacteria (SSB) can play an efficient role in soil by solubilizing insoluble forms of silicates. In addition to this some SSB can also solubilize potassium and phosphates, hence increasing soil fertility and enhancing plant defense mechanisms. A total of 111 bacterial strains were isolated from various habitats of Pakistan and screened for solubilization of silicate, phosphate and potassium on respective media. Out of these, 35 bacterial isolates were capable of solubilizing either silicate, phosphate or potassium. Amongst these 7 bacterial isolates were capable of solubilizing all three minerals tested. The highest silicate (zone diameter 54 mm) and phosphate solubilization (zone diameter 55 mm) was observed for bacterial isolate NR-2 while the highest potassium solubilization was observed for NE-4b (zone diameter 11 mm). Dual culture antagonistic assays were carried out by using these bacterial isolates against four plant pathogenic fungi Magnaporthae grisae, Rhizoctonia solani, Altarnaria alternata and Macrophomina pheasolina. Mean zone of inhibition of these bacterial isolates against the four pathogenic fungi ranged between 4 mm to 39 mm. The largest zone of inhibition against all four bacterial strains was recorded for bacterial isolate NR-2 followed by NE-4b. These strains will be further investigated for their plant growth promoting activities in the future.

Antiviral Effect of Ribonuclease from <i>Bacillus pumilus</i>against Phytopathogenic Rna-Viruses

Background: Viruses can cause different diseases in plants. To prevent viral infections, plants are treated with chemical compounds and antiviral agents. Chemical antiviral agents usually have narrow specificity, which limits their wide application. Alternative antiviral strategy is associated with the use of microbial enzymes, which are less toxic and are readily decomposed without accumulation of harmful substances. The aim of this work is to study the effect of Bacillus pumilus ribonuclease on various phytopathogenic viruses with specific focus on the ability of enzyme to eliminate them from plant explants in vitro. Materials and methods: Extracellular ribonuclease of B. pumilus is tested as an antiviral agent. To study the antiviral effect of RNase, depending on concentration and the time of application several plant-virus model systems are used. Virus detection is conducted by serological testing and RT-PCR. Results: Bacillus pumilus ribonuclease possesses antiviral activity against plant Rna-viruses RCMV (red clover mottle virus), PVX (Potato Virus X) and AMV (Alfalfa Mosaic Virus). The maximum inhibitory effect against actively replicating viruses is observed when plants are treated with the enzyme in the concentration of 100 ug/ml prior to infection. In case of local necrosis ribonuclease in the concentration of 1 ug/ml completely inhibits the development of RCMV virus on bean plants. The enzyme is able to penetrate plants and inhibit the development of viral infection, inhibiting effect for untreated surfaces decreased on average for 20%. It is also found that B. pumilus ribonuclease protects apical explants of sprouts of potato tubers from PVM and PVS viruses. Conclusion: B. pumilus ribonuclease possesses antiviral activity against plant Rna-viruses and produces viruses-free plants in the apical meristem culture.

Antifungal Activity of Oleoresin and Fractions of Pinus elliottii Engelm and Pinus tropicalis against Phytopathogens

Microorganism resistance to the existing products is yet another difficulty that agriculturalists have to deal with. In this context, the search for new agricultural products that can fight phytopathogens has become increasingly important. Plants have played an important role in this process, because they can serve as a source of new compounds for drug discovery. Plants belonging to the genus Pinus produce an oleoresin that protects the plant against herbivores and pathogens. With a view to developing products that can combat fungal pathogens without harming the environment, this work aimed to evaluate the antifungal activity of the oleoresins and fractions of Pinus elliottii Engelm and Pinus tropicalis against phytopathogens. The methodology based on NCCLS M38-A standards aided antifungal activity assessment. The microdilution method helped to determine the Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC). The oleoresins of P. elliottii and P. tropicalis afforded the most significant results—they displayed fungicidal activity against all the tested species. MIC values were promising, especially the MIC of the oleoresin of P. elliottii against S. rolfsii (1.95 μg·mL-1). The MIC values of the oleoresins of P. elliottii and P. tropicalis ranged from 1.95 to 1000 μg·mL-1 and from 31.25 to 250 μg·mL-1, respectively. Fraction PT2 of P. tropicalis furnished the best results among all the assayed fractions: MIC values lay between 125 and 500 μg·mL-11. In conclusion, the oleoresin of P. tropicalis is a promising source of new antifungal agents for application in the treatment of phytopathogenic infections.

Release of active oxygen from phytopathogenic bacteria and their regulation

Some phytopathogenic bacteria including Xanthomonas oryzae pv. oryzae （a rice leaf bacterial blight pathogen） and its virulent gene mutant XooM3105,Xanthomonas campestris pv. campestris, Erwinia carotovora subsp, carotovora and so on, could release active oxygen such as O2 and H2O2 by themselves.Ca2+, NADPH and O2 concentration regulated the response. The response is sensitive to heat and proteinase. These results show that the component releasing active oxygen in the bacterial plant pathogen may involve special protein and enzyme and be secreted to culture liquid. Various components of rice tissue can significantly induce the release of active oxygen in Xanthomonas oryzae pv. oryzae.

Reprogramming of phytopathogen transcriptome by a non-bactericidal pesticide residue alleviates its virulence in rice

Bacteria equipped with virulence systems based on highly bioactive small molecules can circumvent their host's defense mechanisms.Pathogens employing this strategy are currently threatening global rice production.In the present study,variations in the virulence of the highly destructive Barkholderiaplantarii were observed in different rice-producing regions.The environment-linked variation was not attributable to any known host-related or external factors.Co-occurrence analyses indicated a connection between reduced virulence and 5-Amino-l,3,4-thiadiazole-2-thiol(ATT),a non-bactericidal organic compound.ATT,which accumulates in rice plants during metabolization of specific agrochemicals,was found to reduce virulence factor secretion by B.plantarii up to 88.8%and inhibit pathogen virulence by hijacking an upstream signaling cascade.Detailed assessment of the newly discovered virulence inhibitor resulted in mechanistic insights into positive effects of ATT accumulation in plant tissues.Mechanisms of virulence alleviation were deciphered by integrating high-throughput data,gene knockout mutants,and molecular interaction assays.TroK,a histidine protein kinase in a two-component system that regulates virulence factor secretion,is likely the molecular target antagonized by ATT.Our findings provide novel insights into virulence modulation in an important plant-pathogen system that relies on the host's metabolic activity and subsequent signaling interference.

Design, synthesis and SAR study of novel sulfonylurea derivatives containing arylpyrimidine moieties as potential anti-phytopathogenic fungal agents

Acetohydroxyacid synthase(AHAS) was considered as a promising target for antifungal agents.Herein,three series of novel sulfonylureas(SUs) 9-11 containing aromatic-substituted pyrimidines were designed and synthesized according to pharmacophore-combination and bioisosterism strategy.The in vitro fungicidal activities against ten phytopathogenic fungi indicated that most of the title compounds exhibited broad-spectrum and excellent fungicidal activities.Based on the preliminary fungicidal activities,a CoMFA model was constructed and the 3 D-QSAR analysis indicated that either a bulky group around the 5-position of the pyrimidine ring or electropositive group around the 2-position of the benzene ring would be favour to fungicidal activities.In order to study interaction mechanism,10 k was automatically docked into yeast AHAS and it further indicated that bearing bulky groups-aryl at the pyrimidine ring was critical to enhance antifungal activities.It revealed that the antifungal activity of derivatives 9-11 probably results from the inhibition of fungal AHAS.Thus,the present results strongly showed that SUs should be considered as lead compounds or model molecules to develop novel antiphyt o pathogenic fungal agents.

Discovery of cryptolepine derivatives as novel promising agents against phytopathogenic bacteria

To ensure the production of food crops,a series of cryptolepine derivatives were synthesised,after which their antibacterial activities and mechanism of action against three plant pathogens were investigated.Our bioassay results indicated that most of the target compounds displayed potent inhibitory effects against Xanthomonas oryzae(X.oryzae)and Xanthomonas axonopodis pv.citri(X.axonopodis pv.c.).Remarkably,compound 9 exhibited the best in vitro antibacterial activity against X.oryzae,with a minimum inhibitory concentration(MIC)value of 0.78μg·mL^(-1).Compound 2 exhibited the best in vitro antibacterial activity against X.axonopodis pv.c.,with an MIC value of 0.39μg·mL^(-1).These activities were superior to those of copper quinolate(MIC=6.25,25μg·mL^(-1))and thiodiazole copper(MIC=100,200μg·mL^(-1))against X.oryzae and X.axonopodis pv.c.In vivo experiments demonstrated the promising applicability of compound 9 for the control of rice bacterial infections.Furthermore,compound 9 was selected as a candidate to conduct preliminary analyses of the antibacterial mechanisms of cryptolepine derivatives.Scanning electron microscopy and transmission electron microscopy observations,extracellular polysaccharide production,biofilm formation,transcriptomic,quantitative reverse transcription-polymerase chain reaction analyses,and molecular docking assays were performed.Collectively,our findings demonstrated that compound 9 might act via multifarious mechanisms to down-regulate virulence factors and cause cell death.

石榕树中补骨脂素分离鉴定及其抗真菌活性

[目的]分离出石榕树中能够有效抑制植物病原真菌的活性单体化合物并探索其抗真菌活性。[方法]采用生物活性追踪和化学分离相结合的方法对石榕树甲醇提取物进行分离,运用质谱、核磁共振氢谱和碳谱并结合相关文献数据鉴定活性单体化合物。采用菌丝生长速率法测定活性单体化合物对7种植物病原真菌的抑菌活性。[结果]石榕树(叶)甲醇提取物对甘蔗凤梨病菌具有良好抑菌活性,在10 mg·mL^(-1)浓度下,抑菌率为100%。在浓度为1.5 mg·mL^(-1)时,石榕树(叶)石油醚、乙酸乙酯、正丁醇萃取物和水层萃余物对甘蔗凤梨病菌的抑菌率分别为58.73%、100.00%、39.49%和6.63%。表明石榕树(叶)乙酸乙酯层萃取物对甘蔗凤梨病菌的抑制作用最高,其EC_(50)值为0.430 7 mg·mL^(-1)。石榕树(叶)乙酸乙酯层萃取物进行柱色谱分离后得到对甘蔗凤梨病菌有较高抑菌活性的是LMNO1bc1a活性组分,0.1 mg·mL^(-1)的LMNO1bc1a活性组分对甘蔗凤梨病菌的抑菌率为100.00%。LMNO1bc1a组分经结晶、重结晶纯化后命名为SRS-1,SRS-1经NMR和MS技术鉴定为补骨脂素(psoralen)。SRS-1对甘蔗凤梨病菌、甘蓝黑斑病菌、玉米大斑病菌、罗汉果白绢病菌、柑橘砂皮病菌、茶轮斑病菌、烟草黑胫病菌这7种植物病原真菌都有一定的抑菌活性,其EC_(50)值分别为0.018 2、0.038 8、0.040 9、0.051 2、0.080 7、0.092 7、0.115 4 mg·mL^(-1)。[结论]从石榕树中分离出抑菌活性单体化合物SRS-1,鉴定为补骨脂素,其对甘蔗凤梨病菌、甘蓝黑斑病菌、玉米大斑病菌、罗汉果白绢病菌、柑橘砂皮病菌、茶轮斑病菌、烟草黑胫病菌这7种植物病原真菌都有一定的抑菌活性。

微生物除草剂的研究进展

杂草对农业生产构成了巨大的威胁,对农业经济带来了深远的影响。过度依赖化学除草剂的现象引发了大量的抗药性杂草种群出现,这不仅增加了除草剂的使用频率和用量,还会引发恶性循环。通过对现有文献和研究数据的分析,阐述了微生物除草剂的分类及其对目标杂草物种的生物除草活性和特异性。微生物除草剂凭借其延缓杂草产生耐药性、对生态环境影响较小、安全性高和资源充足等独特优势,有望满足可持续农业发展的需求。本文进一步分析了国内外微生物除草剂的发展现状及面临的限制因素,并展望其未来的发展方向和利用趋势。

以粪污水热资源化为核心的高效种养结合系统分析与展望

种养结合系统是实现养殖业和种植业可持续发展的重要途径。水热技术可以将畜禽粪污快速直接转化生产生物原油、水热炭、水相产物和气体产物,生成物分别具有燃料、土壤改良、农用杀菌剂和气体肥料应用潜力,以粪污水热资源化为核心构建的高效种养结合系统有助于提升畜禽粪污的无害化资源化水平和消纳能力、构建生态可持续的绿色循环农业,实现畜禽粪污资源化利用、种植业和养殖的协同高效发展。该研究基于国内外近十余年的研究,系统综述了粪污水热资源化产物在种养结合生态循环农业模式中的作用和应用潜力,论述了畜禽粪污水热转化生物原油在农业内燃机中作为燃料的研究现状,阐述了水相产物作为潜在的农用杀菌剂在作物病害防控中的作用,梳理了畜禽粪污源水热炭在转化机理、理化特性和还田应用中的研究进展,分析了气相产物中的组分和用于温室种植的潜力。在此基础上总结了以粪污水热资源化为核心的高效种养结合系统面临的主要挑战,并对水热资源化产物农业循环应用的研究方向进行展望。研究为畜禽粪污水热资源化和高效种养结合系统研究提供理论参考。

菌毛在生防细菌定殖植物病原线虫中功能的研究进展

植物病原线虫对林业生产危害严重,缺乏有效的防控措施,已造成巨大的经济损失。生防细菌已被用于林木病原线虫的绿色防控,并已取得良好的防治效果。然而,生防细菌防控林木线虫病害的作用机制尚不明确,严重阻碍了防线虫微生物制剂的研发与高效应用。目前,已有大量的研究结果证实,生防细菌在线虫上的定殖能力与其杀线防病效果密切相关,且细菌菌毛在生防细菌于线虫宿主的定殖过程中发挥着重要功能。本文综述了革兰氏阴性细菌菌毛的种类以及菌毛在生防细菌定殖线虫中的功能,讨论了生防细菌菌毛对运动性、黏附性、生物膜形成等定殖相关生物学性状的影响,并对菌毛在细菌防控林木线虫病害中功能的研究与应用进行了展望。

N-芳基氨基甲酸苄酯的合成及抗真菌活性研究

为发现更多抗真菌药物分子,设计合成16种N-芳基氨基甲酸苄酯类目标化合物,并进行结构鉴定;以醚菌酯为阳性对照,采用菌丝线性生长速率法,测定目标化合物对常见的10种植物病原真菌的体外抑制活性,以及5种高抗菌活性的目标化合物对苹果轮纹病原菌和番茄早疫病原菌的毒力。结果发现,所有目标化合物对10种供试真菌都有不同程度的抑制活性,其中,化合物B11抗菌活性最高,对9种供试真菌的抑制率均超过80%,对马铃薯干腐病原菌的抑制率为98.1%,对番茄早疫病原菌的EC50为7.6μg/mL。构效关系研究表明,-F和-Me的引入有利于提高活性,是该类化合物的优势基团;-F和-Me的组合可大幅度提高活性,是优势基团叠加效应的结果。

葡萄酸腐病绿色防控技术

酸腐病是葡萄生产上为害较大的病害之一,是一种由真菌、细菌和果蝇共同为害形成的二次侵染性病害。本文总结了葡萄酸腐病的症状,分析了引起该病的主要病原菌,包括曲霉属、假丝酵母属、有孢汉逊酵母属、伊萨酵母属和醋酸杆菌属,以及该病害的主要传播介质黑腹果蝇、斑翅果蝇和叔白颜果蝇的生物学特性及其对酸腐病发生的影响,并集成了农业防治、物理防治、生物防治及化学防治绿色防控技术。在此基础上,分析了该病害的绿色防控策略路径,旨在为酸腐病绿色防控及葡萄绿色规范生产提供参考。

新型吡唑乙酰胺类琥珀酸脱氢酶抑制剂的设计、合成及抑菌活性

为开发出新型杀菌剂候选分子,通过柔性改造吡唑甲酰胺杀菌剂结构中的二元酰胺键得到了一系列潜在靶向真菌琥珀酸脱氢酶(SDH)的新型吡唑乙酰胺分子.借助菌丝生长速率法发现了其中具有广谱抑菌特性的二苯醚联吡唑乙酰胺分子(6l),其在药剂质量浓度为50μg/mL时对水稻纹枯病菌、小麦赤霉病菌和草莓灰霉病菌的抑制效果均优于对照药剂噁霉灵.化合物6l对水稻纹枯病菌的半最大效应浓度(EC_(50)值)为19.92μg/mL,抑菌活性明显优于对照药剂噁霉灵和氟吡菌酰胺(EC_(50)分别为76.74和40.36μg/mL).SDH酶活力测试结果表明,真菌体内的SDH是化合物6l抑制水稻纹枯病菌生长的潜在作用靶标.分子对接研究结果显示,化合物6l分子结构中的二苯醚单元能以多种方式与靶标口袋的关键残基结合,对分子发挥抑菌活性起到了关键作用.研究结果表明,二苯醚联吡唑乙酰胺分子对植物病原真菌具有较显著的抑制作用,在其结构基础上进行深度的优化和改造有望得到可有效防控植物病原真菌的新型杀菌剂候选分子.

沼液对植物病害的防治效果及机理研究Ⅰ:对植物病原真菌的抑制效果及抑菌机理初探

采用菌丝生长抑制和分生孢子萌发抑制的试验方法,以草莓枯萎病菌为靶标病原菌,研究了江苏省内运行稳定的21个大型沼气工程的沼液及其无菌滤液对草莓枯萎病菌的生长抑制特性。结果表明,所有沼气工程的沼液对草莓枯萎病菌的菌丝生长都有不同程度的抑制作用,不同运行年限的沼气池沼液以及不同发酵原料的沼液对草莓枯萎病菌的抑制效果均无显著差异,但不同贮液阶段的沼液对其抑菌效果影响显著。沼液的无菌滤液对草莓枯萎病菌菌丝的生长没有抑制作用;沼液及其无菌滤液对草莓枯萎病菌分生孢子的萌发均有不同程度的抑制作用,猪粪沼液及其无菌滤液对草莓枯萎病菌分生孢子萌发的抑制作用好于牛粪沼液。沼液对生产上常见的5种植物病原真菌的抑制效果有显著差别。沼液中的拮抗微生物是沼液抑菌防病的主要因子。

稀土积累对植物病原细菌生长的影响

本文研究了单一稀土氧化镧（Ｌａ２Ｏ３）在浓度为５０，１００，１５０，２００，２５０，３００，３５０，４００，５００，６００，７００ｐｐｍ时对植物病原细菌（Ｅｒｗｉｎｉａｃｈｒｙｓａｎｔｈｅｍｉ，Ｅｃｈ，Ｘａｎｔｈｏｍｏｎａｓｏｒｙｚａｅｐｖ．ｏｒｙｚａｅ，Ｘｏｏ；Ｐｓｅｕｄｏｍｏｎａｓｓｏｌａｎａｃｅａｒｕｍ，Ａｚ；Ｃｌａｖｉｂａｃｔｅｒｍｉｃｈｉｇａｎｅｎｓｅｓｕｂｓｐ．ｍｉｃｈｉｇａｎｅｎｓｅ，Ｃ３）生长的影响。在平板培养基中，当稀土浓度大于１００ｐｐｍ时，Ｅｃｈ，Ｘｏｏ，Ａｚ三种细菌菌落出现时间均比对照延迟，Ｃ３在所有处理中不生长。稀土浓度越大，菌落出现时间越迟，在５０～３５０ｐｐｍ内，不同菌落出现时间延迟１２～４８小时。随着稀土浓度提高，生长量逐渐减少，至４００ｐｐｍ时，所有菌不生长。在液体培养基中，当稀土浓度小于２００ｐｐｍ，培养时间为２４小时时，可刺激Ｅｃｈ菌的生长。除此之外，不同稀土浓度不同培养时间下，稀土对植物病原细菌均有一定的抑制作用，抑制作用随稀土浓度提高而增加，至３５０ｐｐｍ时，几乎完全抑制病菌生长，其中对Ｃ３的抑制作用最明显。

植物病原菌拮抗性野生艾蒿内生菌的分离、筛选和鉴定

从野生艾蒿的根茎叶部位分离出内生细菌共68株,以棉花枯萎病、稻瘟病、烟草赤星为供试病原菌,采用对峙法对内生菌分别进行抑菌试验,对筛选出的菌株进行了16S rDNA序列测定和系统发育分析。结果表明:从野生艾蒿分离到的内生菌经初筛、复筛,获得抑制病原菌效果最明显的3株菌,结合生理生化特性、菌落特征、细胞形态特征和16S rDNA测序分析结果,菌株L8、S11和R6分别鉴定为Bacillus subtilis,Bacillus cereus,Paenibacillus polymyxa。拮抗实验表明,棉花枯萎病原菌菌丝发生弯曲、打结,烟草赤星的受作用菌丝生长端分枝明显增多,生长端边缘呈珊瑚状分枝,并且出现明显的畸形和萎缩现象。分析表明,可能是由于在培养过程中内生菌产生了化感物质,对病原菌的菌丝产生抑制作用的结果。

rDNA-ITS在植物病原真菌分子检测中的应用

文章综述了rDNA序列结构特点及ITS序列在植物病原真菌的分类鉴定、分子检测、病害诊断及土壤中病原真菌的检测方面的应用,并对其应用前景进行了评述。