枯草芽孢杆菌(Bacilllus subtilis)S9对植物病原真菌的溶菌作用

本研究共分离了976株细菌分离物,发现来自甘蔗根围的1株枯草芽孢杆菌(Bacillus subtilis)S9对立枯丝核菌(Rhizoctonia solani)、终极腐霉(Pythium ultimum)和西瓜枯萎病菌(Fusarium oxysporum f.sp.niveum)在PDA平板的对峙培养过程中不形成抑菌圈,但4 d后可使上述植物病原真菌的菌丝溶解.扫描电镜观察发现S9菌株在待测的立枯丝核菌表面形成了溶菌斑.S9菌株对立枯丝核菌的作用过程是通过吸附在病原真菌的菌丝上,并随菌丝生长而生长,而后产生溶菌物质消解菌丝体.液体共培养测定也证明了S9菌株具有上述作用.本研究还发现,S9菌株对植物病原真菌的拮抗真菌绿色木霉(Trichoderma viride)、鲜红毛壳菌(Chaetomium cupreum)和球毛壳菌(Chaetomiumglobosum)的生长没有影响.盆栽试验证明了S9菌株可有效地控制立枯丝核菌(R.solani)引起的番茄苗期病害.S9菌株与其它拮抗真菌混合具有促进防治植物病原真菌引起的植物病害的潜力.     

一株拮抗4种马铃薯病原真菌的拟康宁木霉的分离鉴定

本研究从黑龙江省土壤中分离获得一株木霉, 运用生物学特征和ITS序列分析相结合的方法将其鉴定为拟康宁木霉Trichoderma koningiopsis?薯块活体抑菌测定表明, 该木霉对接骨木镰孢Fusarium sambucinum?马铃薯早疫病菌Alternaria solani?立枯丝核菌Rhizoctonia solani (AG3和AG5)等病原真菌引起的马铃薯病害具有抑制作用?对峙培养试验结果显示, 该木霉对上述病原菌的生长具有拮抗作用, 平均抑制率均超过50%, 且该木霉生长速度快于上述病原真菌, 以竞争生长方式抑制病原菌菌丝生长和菌核形成?采用扫描电子显微镜观察发现, 该木霉可通过缠绕或并行生长等方式寄生在上述病原真菌菌丝上?以上结果表明, 从土壤中分离获得的拟康宁木霉具备良好的生防潜力, 可为马铃薯种植中多种真菌病害的统防统治提供良好的生防菌株?     

木霉对土传病原真菌的拮抗作用

 分别在体外及温室测定了筛选菌株哈茨木霉Trichoderma harzianum(T₈₂)和Tricho-derma sp.(NF9)对土传病原真菌的拮抗作用。体外测定表明,木霉菌株T₈₂和NF₉对白绢病菌Sclerotium rolfsii,立枯丝核菌Rhizoctonia solani,瓜果腐霉Pythium aphanidermatum刺腐霉P.spinosum和尖镰孢Fusarium oxysporum在对崎培养中的拮抗系数分别为2或2~3和2。温室测定表明,用0、6%(W/W)T₈₂麸皮培养物(10⁷cfu/g)处理土壤。在人工接种白绢病菌,立枯丝核菌及瓜果腐霉20天后,黄瓜发病率分别比未用木霉处理的对照减少46、5%,28.4%和81。2%;用T₈₂和NF₉木霉孢子悬浮液(10⁸cfu/ml)处理黄瓜种子,人工接种白绢病菌11天后,黄瓜成苗率分别比未用木霉处理的对照增加14%的20%。分别在光学显微镜和扫描电镜下观察到木霉T₈₂对白绢病菌菌丝和菌核的重寄生以及木霉T₈₂和NF₉对立枯丝核菌菌丝的缠绕。穿入及寄生。作者认为重寄生可能是试验木霉菌株T₈₂和NF₉对白绢病菌和立枯丝核菌的主要拮抗机制。     

灰黄青霉CF3对马铃薯土传病原真菌的拮抗性及其促生作用

为探索灰黄青霉Penicillium griseofulvum CF3对马铃薯土传病害病原真菌立枯丝核菌Rhizoctonia solani、茄病镰刀菌Fusariumsolani、硫色镰刀菌F.sulphureum及大丽轮枝菌Verticillium dahliae的拮抗性及对马铃薯的促生作用,采用菌丝生长速率法和甜瓜种子发芽法分别研究了CF3发酵液对病原菌菌丝的抑制作用、对立枯丝核菌微菌核形成的影响及其发酵液的促生作用,并采用盆栽试验法研究了CF3孢子粉对马铃薯植株的促生作用及对抗逆性的影响.CF3发酵滤液对4株病原真菌菌丝生长的抑菌率达53％～72.1％,对立枯丝核菌微菌核的抑制率达36.8％～100％,并显著促进甜瓜种子胚根、胚轴生长.灰黄青霉孢子粉拌土和包衣接种均能促进马铃薯植株生长并增强植株的抗逆性.其中,拌土接种使马铃薯地上植株鲜重和多酚氧化酶活性较对照分别增加38.3％和9％,丙二醛含量降低28.8％.研究表明灰黄青霉CF3对连作马铃薯常见土传真菌病害有较强的生防潜力,对马铃薯具有良好的促生作用.     

草莓丝核菌根腐病病原菌鉴定及7种杀菌剂的抑菌作用测定

为了解草莓丝核菌根腐病的病原种类及筛选防治丝核菌根腐病的有效杀菌剂,本研究基于形态学特征、细胞核荧光染色、菌丝融合群测定以及rDNA-ITS的序列分析,对北京和河北承德地区的草莓丝核菌根腐病的病原菌进行了鉴定,并利用菌丝生长速率法测定了7种杀菌剂对丝核菌的抑菌作用。结果发现,北京地区的丝核菌为双核丝核菌(binucleate Rhizoctonia,BNR),属于融合群AG-A;河北的丝核菌为立枯丝核菌Rhizoctonia solani,属于融合群AG-4。氟啶胺、吡唑醚菌酯、噻呋酰胺、戊唑醇、咯菌腈、氟硅唑对2种丝核菌均有很强的抑制作用,EC_(50)值为0.063 9～2.485 7μg/mL,抑霉唑的抑制作用较差,EC_(50)值为9.966 8～11.236 8μg/mL。同一种杀菌剂对不同丝核菌的抑制作用存在差异,噻呋酰胺、戊唑醇、氟硅唑、咯菌腈和抑霉唑对立枯丝核菌的抑制作用强于对双核丝核菌。试验结果为生产上合理选用杀菌剂防治草莓丝核菌根腐病提供了科学依据。     

马铃薯立枯丝核菌生物学特性研究

采用生长速率法研究了黑龙江省马铃薯立枯丝核菌(Rhizoctonia solani Kühn)菌丝生长的条件。结果表明病菌菌丝在10~35℃均能生长,适宜温度是25~30℃,30℃菌丝生长最快。在pH为3~12的范围内病菌菌丝均能生长,以pH 5~7菌丝生长速度较快。黑暗对菌丝生长有促进作用,光照可促进菌核的形成。不同碳源、氮源对立枯丝核菌菌丝生长的影响均较明显,可溶性淀粉和酵母粉对菌丝生长有显著的促进作用。立枯丝核菌菌丝体及菌核的致死温度分别为49℃和51℃。明确了黑龙江省马铃薯立枯丝核菌生长的环境条件,可为病害防治提供理论依据。     

8种植物精油对4种植物病原真菌的抑菌活性及椒蒿精油的GC-MS成分分析

为探究植物精油对4种植物病原真菌的抑菌活性,通过水蒸汽蒸馏法提取8种植物精油,菌丝生长抑制法测定精油对尖孢镰刀菌、串珠镰刀菌、黄色镰孢菌和立枯丝核菌的抑菌活性。结果表明：驱虫斑鸠菊精油无抑菌活性,其他7种植物精油均有一定的抑菌活性,其中香青兰和椒蒿精油的活性最强,在体积浓度为0.5 µL/mL时,香青兰精油对尖孢镰刀菌、黄色镰孢菌和立枯丝核菌的抑菌率在95%以上,椒蒿精油对4种病原真菌的抑菌率在70%以上。在体积浓度为0.05 µL/mL时,椒蒿精油对4种病原真菌的抑菌率均在50%以上,香青兰精油对尖孢镰刀菌、黄色镰孢菌和立枯丝核菌的抑菌率在35%以上。通过气相色谱−质谱联用（Gas Chromatography−Mass Spectrometer, GC−MS）技术对椒蒿精油进行化学成分分析,a,a-二甲基-1,4-二氧-2-奈乙醛的相对含量最高,占比72.17%。本研究可为植物病原微生物的绿色防控提供基础资料。     

南方红豆杉内生真菌的抗菌活性

从南方红豆杉中分离到549株内生真菌,以终极腐霉Pythiumultimum、立枯丝核菌Rhizocto-niasolani为靶标菌进行抗菌活性的筛选。结果表明,对终极腐霉、立枯丝核菌的半抑制稀释倍数(ID50)在10以上的活性菌株分别为20株(占总菌株的3.6%)和15株(占总菌株的2.8%)。其中矮棒曲霉Aspergillusclavatonanicus菌株F0028对这两种病原菌的活性最高(对终极腐霉、立枯丝核菌的ID50分别为278±15.0、108±18.6)。进一步研究表明,F0028发酵液在pH值为1.0~7.0之间均有较强的抑菌活性;经高温高压处理后抑菌活性仍达到了对照的(65±3.8)%。发酵液经高效液相色谱纯化得到5种化合物,活性测定表明,5种化合物对终极腐霉、立枯丝核菌均有不同程度的抑制作用,半抑制浓度(EC50)在7.0~45μg/mL之间。     

球毛壳ND35在杨树的定植及对酶活性的影响

球毛壳Chaetomium globosum ND35菌株是一株分离自健康毛白杨的内生菌菌株,能对多种植物病原真菌产生明显的拮抗作用[1],但其侵染的方式及侵染后对植物的影响未见报道.     

哈茨木霉Thga1基因敲除突变株对几种植物病原真菌的离体拮抗作用

Thga1是哈茨木霉Th-33的1种I型G蛋白α亚基基因(GenBank JN874387)。本文针对前期研究获得的2株基因Thga1敲除突变株ΔThga1 1-1和ΔThga1 10-1,通过平皿对峙培养分析其对立枯丝核菌、辣椒疫霉菌和尖孢镰刀菌的拮抗作用,研究了突变株产生的挥发性代谢物和非挥发性代谢物对上述3种病原菌生长的影响和对立枯丝核菌的重寄生作用的光学显微镜观察。结果表明,与野生型Th-33相比,基因敲除突变株ΔThga1 1-1和ΔThga1 10-1对病原菌的拮抗作用显著下降,不能对3种病原菌菌落进行覆盖和寄生;突变株产生的挥发性代谢产物对病原菌生长的抑制率显著低于野生菌Th-33,2株突变株对立枯丝核菌、辣椒疫霉和尖孢镰刀菌生长的抑制率分别降低了50%~57%、43%~58%和58%~63%,且突变株之间差异不显著;突变株产生的非挥发性代谢产物对病原菌生长的抑制作用中,除突变株ΔThga1 1-1对立枯丝核菌的抑制作用与野生菌相比差异不显著外,均显著低于野生菌Th-33,突变株ΔThga1 1-1和ΔThga110-1对3种病原菌的抑制率分别降低了8%~25%、23%~56%和38%~50%。显微观察发现,野生菌Th-33菌丝能够缠绕、附着、穿透和裂解立枯丝核菌菌丝,但突变株菌丝对立枯丝核菌菌丝没有发现类似作用。上述结果表明,Thga1基因影响了哈茨木霉菌的拮抗、次级代谢和重寄生等作用。     

一株生防内生真菌的分离筛选、鉴定及抑菌特性

为获取优良植物病害生防菌,以交链孢菌Alternaria tenuis Ness和尖镰孢菌Fusarium oxysporum Schlecht为指示菌,采用平板对峙培养法、抑菌圈法和菌落直径法对健康龙柏鳞叶小枝中分离的4株优势内生真菌进行了筛选评价,并结合形态学观察及ITS序列分析对筛选出的生防内生真菌进行了鉴定。结果表明,对峙培养发现1菌株对交链孢菌和尖镰孢菌均具有较好的抑制作用,抑菌率分别为52.05%和48.65%,标记其为LB-1;LB-1菌株与指示菌对峙培养的菌落交界面处,2种指示菌的菌丝均出现了消解、断裂,而LB-1的菌丝则生长正常。未灭菌LB-1菌株发酵液对交链孢菌和尖镰孢菌产生的抑菌圈直径分别为14.1 mm和13.7 mm,抑菌率分别为47.89%和48.72%;灭菌LB-1菌株发酵液对交链孢菌和尖镰孢菌的抑菌圈直径分别为15.3 mm和15.0 mm,抑菌率分别为5.96%和33.90%,指示菌在生长过程中菌丝溶解现象明显。经鉴定,LB-1菌株为毛壳菌属Chaetomium中的近缘毛壳菌C.subaffine,是一种极具生防潜力的内生真菌,其抑菌特性可能通过溶菌作用实现。     

黑龙江省马铃薯早疫病菌生物学特性研究

[目的]明确黑龙江省马铃薯早疫病菌(Alternaria solani)生物学特性的多样性.[方法]通过平板培养研究分离自黑龙江省马铃薯早疫病菌菌株的生物学特性.[结果]各分离株在PDA培养基上菌落生长速度、颜色、形态、成带现象等差异较大.其中的5个代表性供试菌株在PDA、CMA、OA培养基上生长较好,均能在10～35℃生长,但最适温度有一定的差异;供试菌株均能在pH 4～10的条件下生长,最适pH为6～8;光照对各分离株菌落生长有促进作用,各菌株对pH、光的反应不完全一致.[结论]说明黑龙江省马铃薯早疫病菌的生物学特性具有明显的遗传多样性.     

立枯丝核菌侵染玉米的研究

 用获自水稻及玉米的立枯丝核菌(Rhizoctonia solani Khn) AG-11A接种玉米发生典型纹枯症状,其致病力显著强于AG-4。玉米拔节期,上位叶鞘抗性较强,抽雄及抽丝期抗性减弱,下位叶鞘无论在拔节期或抽雄、抽丝期,均较上位叶鞘感病。接种玉米后8小时,形成侵染垫及附着胞,从这些结构上形成侵入钉侵入,AG-4侵染上位叶鞘时,常以菌丝直接穿透表皮或从气孔侵入。在去掉菌体的叶鞘表面,发现有周边光滑或稍破损的侵入孔。接种后12小时,在叶鞘细胞中发现菌丝,它们在穿过细胞壁进入邻近细胞时,明显变细。接种后16小时,新生出的菌丝从气孔成丛出现。     

Association of Binucleate Rhizoctonia with Soybean and Mechanism of Biocontrol of Rhizoctonia solani

ABSTRACT The association of binucleate Rhizoctonia (BNR) AG-K with soybean and the interaction of BNR, R. solani AG-4, and soybean seedlings were investigated to elucidate the mechanism of biocontrol of R. solani by BNR. Sixty-hour-old seedlings were inoculated and incubated in a growth chamber at 24 degrees C; plants were examined with light microscopy and with scanning and transmission electron microscopy at various times following inoculation. BNR grew over hypocotyls, roots, and root hairs, but only colonized epidermal cells. Hyphae of BNR appeared to attach to the epidermis and, 5.5 h following inoculation, began penetrating cells by means of penetration pegs without forming distinct appressoria or infection cushions. There was evidence of cuticle degradation at the point of penetration. Infection hyphae moved to adjacent epidermal cells by direct penetration of epidermal radial walls. There were epidermal and cortical cell necrosis, beginning with the fragmentation of the tonoplast and followed by the disintegration of cytoplasm, organelles, and plasma membranes. Cell necrosis was also observed in adjacent cells where there was no evidence of BNR hyphae. Cell walls were not destroyed. After 144 h, there was noevidence of BNR hyphae in cortical cells. Attempted penetrations were observed, but papillae formed on the inside of cortical cell walls. Pre-inoculation of soybean seedlings with BNR 24 or 48 h before inoculation with R. solani (1 cm between inocula) affected the growth of R. solani on soybean tissue. There were fewer hyphae of R. solani, the hyphae branched sparingly, and infection cushions were rare when compared with hyphal growth on soybean inoculated only with R. solani. These effects were observed before the BNR hyphae began to intermingle with the hyphae of R. solani on the surface of the inoculated host. Preinoculation of soybean seedlings 24 h before inoculation with R. solani significantly (P = 0.05) reduced disease incidence and severity caused by R. solani AG-4. The lesions caused by R. solani always appeared distally, not proximally, to the BNR inoculum. The interactions of intermingling hyphae of BNR and R. solani were examined in vitro and on the surface of the host. There was no evidence of lysis, mycoparasitism, inhibition of growth, or any other form of antagonism between hyphae. The results of these studies strongly suggest that induced resistance is the mechanism of biocontrol of R. solani on soybean by BNR. The inhibition of hyphal growth of R. solani on the surface of soybean tissue preinoculated with BNR appears to be a novel characteristic of induced resistance.     

Ultrastructural and Cytochemical Aspects of the Interaction Between the Mycoparasite Pythium oligandrum and Soilborne Plant Pathogens

ABSTRACT The interaction between the oomycete Pythium oligandrum and various soilborne oomycete and fungal plant pathogens (P. ultimum, P. aphanidermatum, Fusarium oxysporum f. sp. radicis-lycopersici, Verticillium albo-atrum, Rhizoctonia solani, and Phytophthora megasperma) was studied by light and electron microscopy in order to assess the relative contribution of mycoparasitism and antibiosis in the antagonistic process. Scanning electron microscope investigations of the interaction regions showed that structural alterations of all pathogenic fungi and oomycetes (except for Phytophthora megasperma) occurred soon after contact with the antagonist. Light and transmission electron microscope studies of the interaction region between the antagonist and P. ultimum revealed that intimate contact between both partners preceded a sequence of degradation events including aggregation of host cytoplasm and penetration of altered host hyphae. Localization of the host wall cellulose component showed that cellulose was altered at potential penetration sites. A similar scheme of events was observed during the interaction between P. oligandrum and F. oxysporum f. sp. radicis-lycopersici, with the exception that complete loss of host protoplasm was associated with antagonist invasion. The interaction between P. oligandrum and R. solani resulted in an abnormal deposition of a wall-like material at potential penetration sites for the antagonist. However, the antagonist displayed the ability to circumvent this barrier and penetrate host hyphae by locally altering the chitin component of the host hyphal wall. Interestingly, antagonist cells also showed extensive alteration as evidenced by the frequent occurrence of empty hyphal shells. In the case of Phytophthora megasperma, hyphal interactions did not occur, but hyphae of the plant pathogen were damaged severely. At least two distinct mechanisms appear to be involved in the process of oomycete and fungal attack by P. oligandrum: (i) mycoparasitism, mediated by intimate hyphal interactions, and (ii) antibiosis, with alteration of the host hyphae prior to contact with the antagonist. However, the possibility that the antagonistic process may rely on the dual action of antibiotics and hydrolytic enzymes is discussed.     

苍耳等14种植物对植物病原菌的抑菌活性

以番茄灰霉菌、辣椒丝核菌、黄瓜枯萎菌、黄瓜黑星菌和番茄早疫菌为供试菌,用生长速率法和孢子萌发法对14种植物提取物的离体生物活性进行了测定。结果表明:在供试质量浓度为0.1 g/mL时,苍耳茎叶、百部根、龙葵对辣椒丝核菌和黄瓜枯萎菌的抑制率达到100%;黄花蒿、苦参根、狼毒根对5种供试菌的抑制率均在60%以上。苍耳根、苍耳茎叶、泽漆、苦参根、龙葵、黄花蒿、蒲公英提取物至少对1种供试菌孢子萌发抑制率大于90%。本试验中,苍耳茎叶和根提取物对供试菌的菌丝生长和孢子萌发均显示了很高的抑制作用。     

Aetiology of Rhizoctonia in sheath blight of maize in Sichuan

Rhizoctonia isolates obtained from maize grown in commercial fields in 33 representative counties (or cities) in Sichuan province in China were characterized according to colony morphology, hyphal anastomosis and pathogenicity. Of 141 isolates, 116 were identified as R. solani , 23 as R. zeae and two as binucleate Rhizoctonia . The isolates of R. solani were assigned to four anastomosis groups (AG): AG-1-IA (101 isolates, accounting for 71.6% of the total), AG-1-IB (2, 1.4%), AG-4 (9, 6.4%) and AG-5 (4, 2.8%). The two isolates of binucleate Rhizoctonia belonged to AG-K. On maize, isolates of AG-1-IA caused typical sheath blight symptoms. Lesions produced by isolates of AG-4, AG-5, AG-1-IB and AG-K were darker than those of AG-1-IA. Rhizoctonia zeae usually caused discontinuous lesions with a dark brown margin and a brown centre on the leaf sheaths, as well as ear rot. Isolates of AG-1-IA were the most virulent to maize, with an average lesion length of approximately 15 cm. Isolates of R. zeae produced lesions approximately 12 cm long, while those of AG-4, AG-5, AG-1-IB and AG-K were progressively shorter. On potato dextrose agar (PDA; pH 6.4), the minimum temperature for mycelial growth of R. zeae isolates was 14–18°C, the maximum 38–40°C and optimum 30°C. Isolates of R. zeae did not grow on PDA (28°C) at pH 2.0, the optimum for growth being pH 6.4.     

拮抗放线菌A03的生防作用及其分类鉴定

从京郊菜园土壤中分离到一株拮抗放线菌,编号为A03。为了科学评价该菌株在植物病害生防中的应用潜力,采用平板对峙培养法和温室盆栽防治法测定了其抑菌活性,并通过形态学、化学和分子分类的方法研究其分类地位。结果表明,该菌株对供试的多种植物病原真菌均有抑制作用,其中对果蔬灰霉病菌、番茄早疫病菌、小麦纹枯病菌和瓜果腐霉的抑制作用较强,平板抑菌带宽度达11．0～12．0 mm。温室防病试验结果表明,其发酵上清液对甜椒灰霉病和番茄灰霉病具有良好的生防作用,防治效果分别为84．97%和79．51%。根据其菌株形态特征、培养性状、生理生化特性、细胞壁化学组分及16S rDNA序列分析,将菌株A03归入链霉菌属,并鉴定为吸水链霉菌Strep- tomyces hygroscopicus。     

粉红粘帚霉67-1菌株寄生核盘菌研究

 用诱捕法从海南乐东县菜园土壤中获得一株对核盘菌菌核具有强寄生能力的粉红粘帚霉(Gliocladium roseum)菌株67-1,寄生频率为100%。该菌的PDA平板回接核盘菌菌核,一周后寄生率可达100%。对峙培养发现其对核盘菌有明显的抑菌带。保湿条件下,该菌孢子在24h内成功侵入核盘菌菌核。切片显微观察证明该菌能高效侵染菌核,造成组织溃解。寄生过程中菌核内蛋白组成发生明显变化。这一菌株在22~35℃均能很好生长,菌丝及产孢最适温度为24℃,产孢量大。认为这一菌株具有良好的菌核病生防应用潜力。     

核盘菌重寄生菌链孢粘帚霉HL-1-1菌株的生物学特性研究

 对植物真菌病害优良生防菌株链孢粘帚霉(Gliocladium catenulatum) HL-1-1的生物学特性进行了研究。结果表明,以核盘菌菌核粉为唯一碳源的培养基最适合HL-1-1的菌丝生长,PDA最适合产孢;蔗糖作为碳源、硝酸钾作为氮源、2530℃、pH值5及黑暗等条件适合HL-1-1的菌丝生长;可溶性淀粉作为碳源、牛肉膏作为氮源、25℃、pH值6及光照等条件适合产孢;菌核浸出液对HL-1-1的分生孢子萌发有促进作用,葡萄糖对分生孢子萌发有抑制作用;2530℃及黑暗条件适合孢子萌发,温度低于5℃或高于40℃,孢子不能萌发;孢子致死温度为52℃。