烟草几丁酶β—1.3—葡聚糖酶的抑菌作用

从三个方面对激发子诱导烟草几丁质酶,β－１．３－葡聚糖酶的病理功能进行了测定,结果表明：①两种酶对赤星菌的菌落生长有抑制作用并可抑制孢子萌发,对其它被测病原物抑制作用非常微弱;②两种酶对赤星菌等有明显的攻击作用,直接攻击抱子的反应在３０ｍｉｎ内可见,到１６ｈ前后引起孢子破裂;③扫描电镜观察,酶对寄主生活叶面上的赤星菌有同样攻击作用。     

烟草几丁酶β-1.3-葡聚糖酶的抑菌作用*

从三个方面对激发子诱导烟草几丁质酶,β－１．３－葡聚糖酶的病理功能进行了测定,结果表明：①两种酶对赤星菌的菌落生长有抑制作用并可抑制孢子萌发,对其它被测病原物抑制作用非常微弱;②两种酶对赤星菌等有明显的攻击作用,直接攻击抱子的反应在３０ｍｉｎ内可见,到１６ｈ前后引起孢子破裂;③扫描电镜观察,酶对寄主生活叶面上的赤星菌有同样攻击作用。     

防治烟草赤星病有益内生细菌的筛选及抑菌作用

从健康烟草的叶、茎中分离到302株非病原内生细菌,通过平板对峙培养,筛选出对烟草赤星病菌［Alternaria alternata（Fr.）Keissl］不同致病力的4个代表菌株均有拮抗作用的11个菌株。室内测定其对赤星病菌抑菌带的宽度达5.5～13.2mm;拮抗、防病试验测定,来自叶片内的内生菌株Itb162对赤星病菌有较强和稳定的拮抗作用,对赤星病有52.0%的防病效果。无菌滤液实验表明,拮抗内生细菌Itb162无菌滤液在一定浓度范围内均能有效地抑制菌丝生长,减少孢子萌发,且浓度越高,抑制能力越强。     

短小芽孢杆菌AR03对烟草赤星病菌和白粉病菌的防治

采用对峙培养法、凹玻片法、LB琼脂培养基萌发法测定短小芽孢杆菌AR03对烟草赤星病菌和白粉病菌的抑制作用.结果表明： AR03菌液对2种病菌的菌丝生长和分生孢子萌发均有明显的拮抗作用;对赤星病菌的抑制作用表现为:经AR03菌液原液(3×10⁸cfu·mL^-1)处理的菌丝隔间变短、肿胀且集结成团,内含物聚集,菌丝顶端生长膨大畸形;经该菌液处理的赤星病菌分生孢子不萌发或萌发产生畸形芽管,分生孢子变形、肿大,纵横分隔部分的组织膨胀呈泡状.AR03菌液原液、30倍液和200倍液对白粉病菌分生孢子萌发的平板抑制率分别为100%、91.4%和 69.3%,菌液对分生孢子萌发的破坏作用表现为分生孢子不萌发,细胞肿胀变形、细胞原生质解体或收缩,孢子内、外壁分离,由于原生质外泄,一些分生孢子内部呈中空状.温室防治试验结果表明： 不同浓度AR03菌悬液处理对烟草白粉病的防治效果存在显著差异,第二次药后7和15 d,AR03菌液原液的防治效果分别达到83.8%和90.3%,与对照药剂差异不显著;而100倍稀释液的防效分别为70.0%和73.3%,与对照药剂差异显著.AR03菌株防治白粉病的持效期为30 d以上.     

飞虱虫疠霉分生孢子在桃蚜体壁上的附着与入侵*

用飞虱虫疠霉(Pandora delphacis)初级分生孢子接种桃蚜(Myzus persicae),24 h内定时取样,在扫描电镜下观察孢子的萌发及其对寄主体壁的入侵。结果表明,附着到蚜体表面的孢子在4 h内有30~40%已萌发产生芽管,其中多数为侵染性芽管,少数是呈叉状分枝的营养生长型芽管。具侵染性芽管的孢子部分陷入体壁蜡质层中,显示孢子有分泌物产生并作用于寄主体壁。接种10 h内,侵染性芽管通过顶端膨大的附着胞或直接穿透侵入寄主体壁。到24 h时,产生侵染性芽管的孢子全部侵入寄主体内,寄主体表仅留下少数未萌发的孢子或营养生长型芽管。初级分生孢子在蚜体表面很少产生次级分生孢子,说明桃蚜是适合该菌侵染的寄主。陷入寄主体壁的孢子不因若蚜蜕皮而被去掉,表明该菌对成蚜和若蚜都具有侵染力。     

飞虱虫疠霉分生孢子在桃蚜体壁上的附着与入侵

用飞虱虫疠霉（Pandora delphacis)初级分生孢子接种桃蚜（Myzus persicae),24h内定时取样,在扫描电镜下观察孢子的萌发及其对寄主体壁的入侵。结果表明,附着到蚜体表面的孢子在4h内有30－40％已萌发产生芽管,少数是呈叉状分枝的营养生长型芽管。且侵染性芽管的孢子部分孢入体壁蜡质层中,显示孢子有分泌物产生并作用于寄主体壁。接种10h内,侵染性芽管通过顶端膨大的附着胞或直接穿透入寄主体壁。到24h时,产生侵染性芽管的孢子全部侵入寄主体内,寄主体表仅留下少数未萌发的孢子或营养生长型芽管。初级分生孢子在蚜体表面很少产生次级分生孢子,说明桃蚜是适合该菌侵染的寄主。陷入寄主体壁的孢子不因若蚜蜕皮而被去掉,表明该菌对成蚜和若蚜都具有侵染力。     

条形柄锈菌(小麦条锈病菌)侵染结构体外形成的观察

通过荧光显微镜和扫描电镜分别对条形柄锈菌夏孢子在寄主植物-小麦叶表和非寄主植物-水稻叶表以及小麦穗部和茎秆上的萌发过程进行了观察。结果发现,夏孢子在小麦叶片体表萌发产生芽管后,可依次分化形成气孔下囊、初生菌丝与吸器母细胞;在小麦颖片、稃片及茎秆部位表面,同样可观察到病菌在体外分化形成吸器母细胞;并且在水稻叶片上也观察到病菌侵染结构存在体外分化现象。经荧光染色发现,条形柄锈菌在体外与在小麦组织中形成的侵染结构没有明显的差别。观察结果可为条形柄锈菌侵染结构的离体诱导与调控机理研究提供依据。     

荧光素钠和考马斯亮蓝应用于小麦白粉病菌染色效果比较*

比较了荧光素钠和考马斯亮蓝应用于小麦白粉病菌染色的效果。荧光素钠法中样品处理只需20min.左右,具有直接、快速的特点;荧光指示剂对病菌分生孢子萌发及菌丝生长无抑制作用,主要沉集于活菌体的隔膜和细胞质部位,使病菌产生明显的亮绿荧光和清晰的细胞轮廓,亮绿荧光衰退期为7min.;借助荧光显微镜可以观察病菌在小麦叶表的发展过程,区别活菌体和失活菌体。考马斯亮蓝法包括传统的组织学染色步骤,经过改进后的样品处理过程需要40min.左右;染色后使寄主组织呈现淡蓝色,病菌菌体染成深蓝色;该方法可以观察病菌在小麦叶表和被侵染细胞内部发育形成的结构,包括孢子发育形成的初生芽管、附着胞芽管、成熟附着胞以及在寄主细胞内形成的初生吸器原体、成熟的指状体吸器和次生吸器。     

转GhB301基因烟草的防御酶活性及抗病相关基因表达分析

为了明确棉花ERF-B3亚族转录因子基因GhB301在烟草异位表达后(抗枯萎病中)的功能,该研究以过表达GhB301基因烟草和野生型烟草为材料,采用枯萎病菌孢子悬浮液接菌方法,分析病原菌侵染前后防御酶活性变化以及防卫相关基因的表达变化与抗病性的关系。结果显示:(1)棉花枯萎病菌处理15d后,2个转基因株系烟草叶片黄化程度与野生型相比较轻。(2)棉花枯萎病菌处理后,过表达GhB301转基因烟草和野生型烟草叶片过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)的活性较未接菌对照显著提高,并且酶活峰值出现均早于野生型材料;转基因材料叶片的POD、PAL、PPO活性均在处理3d后达到峰值,而野生型材料叶片的POD、PAL活性在处理5d后才达到峰值。(3)接种棉花枯萎病菌后活性氧相关基因、乙烯(ET)/茉莉酸(JA)途径相关基因、病程相关基因的表达量在转基因株系OE1和OE2中均受到明显影响。研究推测,GhB301在烟草中的异位表达激活了防卫相关基因的表达,提高了防御酶的活性,从而增强了烟草对枯萎病菌的抗性。     

绿僵菌侵染小菜蛾体表过程的显微观察

采用扫描电镜研究了小菜蛾Plutella xylostella体表结构对绿僵菌入侵行为的影响及绿僵菌的侵染过程。结果表明: 绿僵菌孢子在小菜蛾体表萌发后可形成附着胞,寄主体表结构影响形成附着胞的快慢、多少及穿透体壁时芽管长度, 在平缓结构区和刺状结构区比嵴状结构区更易形成附着胞,且芽管较短。在所有结构区,LF68菌株穿透芽管均短于LD65菌株的芽管。接种后7 h,分生孢子在小菜蛾体表开始萌发,LF68与LD65菌株分别于接种后10 h和13 h出现侵染构造穿透体壁。     

Chronopathological Aspects of Disease Incidence in Rice (Oryza sativa L.)

Rice seedlings maintained under uncontrolled glasshouse conditions were inoculated with conidial suspensions of a fungal pathogen, Helminthosporium oryzae, at various times during the 24 h. Significant increase in the percent germination and germ tube length of conidia were observed in the rice samples inoculated at 02:00 and 06:00h. The 24 h temporal variation in leaf temperature was positively correlated with variation in stomatal movements. The results indicate a 24 h rhythm in the behavior of the fungal pathogen on the host in relation to the conditions of the growing environment. In all the inoculated seedlings, the appearance of a large number of brown leaf spots was confined to the light span. Among the plants inoculated, earlier initiation of brown leaf spot appearance, maximum number of leaf spots, and highest disease severity were observed when plants were inoculated at 02:00h. There was a positive correlation between disease severity of the host and in vivo values of percent germination of conidia and germ tube length of the pathogen in plants inoculated between 02:00 and 06:00h. The findings of this study implicate that light intensity and temperature could play a predominant role in controlling disease susceptibility rhythms in plants.     

Chronopathological aspects of disease incidence in rice (Oryza sativa L.)

Rice seedlings maintained under uncontrolled glasshouse conditions were inoculated with conidial suspensions of a fungal pathogen, Helminthosporium oryzae, at various times during the 24 h. Significant increase in the percent germination and germ tube length of conidia were observed in the rice samples inoculated at 02:00 and 06:00h. The 24 h temporal variation in leaf temperature was positively correlated with variation in stomatal movements. The results indicate a 24 h rhythm in the behavior of the fungal pathogen on the host in relation to the conditions of the growing environment. In all the inoculated seedlings, the appearance of a large number of brown leaf spots was confined to the light span. Among the plants inoculated, earlier initiation of brown leaf spot appearance, maximum number of leaf spots, and highest disease severity were observed when plants were inoculated at 02:00h. There was a positive correlation between disease severity of the host and in vivo values of percent germination of conidia and germ tube length of the pathogen in plants inoculated between 02:00 and 06:00h. The findings of this study implicate that light intensity and temperature could play a predominant role in controlling disease susceptibility rhythms in plants.     

cAMP Regulates Infection Structure Formation in the Plant Pathogenic Fungus Magnaporthe grisea

Magnaporthe grisea, the causal agent of rice blast, is one of the most destructive fungal pathogens of rice throughout the world. Infection of rice by M. grisea requires the formation of an appressorium, a darkly pigmented, dome-shaped structure. The germ tube tip differentiates into an appressorium following germination of conidia on a leaf surface. When conidia germinate on growth medium or other noninductive surfaces, the emerging germ tube does not differentiate and continues to grow vegetatively. Little is known about the endogenous or exogenous signals controlling the developmental process of infection structure formation. We show here that a hydrophobic surface was sufficient for the induction of the appressorium. Furthermore, we demonstrate that the addition of cAMP, its analogs (8-bromo cAMP and N6-monobutyryl cAMP), or 3-isobutyl-1-methylxanthine (an inhibitor of phosphodiesterase) to germinating conidia or to vegetative hyphae induced appressorium formation on noninductive surfaces. The identification of cAMP as a mediator of infection structure formation provides a clue to the regulation of this developmental process. Elucidation of the mechanism involved is not only of biological interest but may also provide the basis for new disease control strategies.     

Cloning and overexpression of antifungal barley chitinase gene in Escherichia coli

Plant chitinases are pathogenesis-related proteins, which are believed to be involved in plant defense responses to pathogen infection. In this study, chitinase gene from barley was cloned and overexpressed in Escherichia coli. Chitinase (35 kDa) was isolated and purified. Since the protein was produced as insoluble inclusion bodies, the protein was solubilized and refolded. Purified chitinase exerted broad-spectrum antifungal activity against Botrytis cinerea (blight of tobacco), Pestalotia theae (leaf spot of tea), Bipolaris oryzae (brown spot of rice), Alternaria sp. (grain discoloration of rice), Curvularia lunata (leaf spot of clover) and Rhizoctonia solani (sheath blight of rice). Due to the potential of broad-spectrum antifungal activity barley chitinase gene can be used to enhance fungal-resistance in crop plants such as rice, tobacco, tea and clover.     

Accumulation of HDMBOA-Glc is induced by biotic stresses prior to the release of MBOA in maize leaves

The effects of biotic stresses on the contents of benzoxazinones (Bxs) were investigated in maize leaves. When the causal agent of southern corn leaf blight, Bipolaris maydis, was inoculated on the third leaf, the amount of 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside (HDMBOA-Glc) increased, reaching a maximum level 48 h after inoculation. The inoculation of weakly pathogenic Curvularia lunata and non-pathogenic Alternaria alternata also resulted in accumulation of HDMBOA-Glc, and filtrates of the cultures of B. maydis, C. lunata and A. alternata also showed the accumulation of elicitor-active compounds by the fungi. Furthermore the infection of B. maydis induced formation of dark brown lesions, where most abundant Bx-related compound was 6-methoxy-2-benzoxazolinone (MBOA). The later is formed by degradation of DIMBOA and HDMBOA, whereas HDMBOA-Glc was most abundant in the surrounding green tissues. Among the Bx-related compounds, MBOA exhibited the strongest inhibition of the germination of the conidia and of the growth of germ tubes of B. maydis, C. lunata and A. alternata. In addition to fungal infection, the feeding by rice armyworm larvae resulted in HDMBOA-Glc accumulation. These findings are discussed in relation to the possible ecological relevance of the conversion of DIMBOA-Glc into HDMBOA-Glc.     

Phylloplanins of tobacco are defensive proteins deployed on aerial surfaces by short glandular trichomes

In plants, defensive proteins secreted to leaf aerial surfaces have not previously been considered to be a strategy of pathogen resistance, and the general occurrence of leaf surface proteins is not generally recognized. We found that leaf water washes (LWW) of the experimental plant Nicotiana tabacum tobacco introduction (TI) 1068 contained highly hydrophobic, basic proteins that inhibited spore germination and leaf infection by the oomycete pathogen Peronospora tabacina. We termed these surface-localized proteins tobacco phylloplanins, and we isolated the novel gene T-Phylloplanin (for Tobacco Phylloplanin) and its promoter from N. tabacum. Escherichia coli-expressed T-phylloplanin inhibited P. tabacina spore germination and greatly reduced leaf infection. The T-phylloplanin promoter, when fused to the reporter genes beta-glucuronidase and green fluorescent protein, directed biosynthesis only in apical-tip cell clusters of short, procumbent glandular trichomes. Here, we provide evidence for a protein-based surface defense system in the plant kingdom, wherein protein biosynthesis in short, procumbent glandular trichomes allows surface secretion and deposition of defensive phylloplanins on aerial surfaces as a first-point-of-contact deterrent to pathogen establishment. As yet uncharacterized surface proteins have been detected on most plant species examined.     

Different Chitin Synthase Genes Are Required for Various Developmental and Plant Infection Processes in the Rice Blast Fungus Magnaporthe oryzae

Chitin is a major component of fungal cell wall and is synthesized by chitin synthases (Chs). Plant pathogenic fungi normally have multiple chitin synthase genes. To determine their roles in development and pathogenesis, we functionally characterized all seven CHS genes in Magnaporthe oryzae. Three of them, CHS1, CHS6, and CHS7, were found to be important for plant infection. While the chs6 mutant was non-pathogenic, the chs1 and chs7 mutants were significantly reduced in virulence. CHS1 plays a specific role in conidiogenesis, an essential step for natural infection cycle. Most of chs1 conidia had no septum and spore tip mucilage. The chs6 mutant was reduced in hyphal growth and conidiation. It failed to penetrate and grow invasively in plant cells. The two MMD-containing chitin synthase genes, CHS5 and CHS6, have a similar expression pattern. Although deletion of CHS5 had no detectable phenotype, the chs5 chs6 double mutant had more severe defects than the chs6 mutant, indicating that they may have overlapping functions in maintaining polarized growth in vegetative and invasive hyphae. Unlike the other CHS genes, CHS7 has a unique function in appressorium formation. Although it was blocked in appressorium formation by germ tubes on artificial hydrophobic surfaces, the chs7 mutant still produced melanized appressoria by hyphal tips or on plant surfaces, indicating that chitin synthase genes have distinct impacts on appressorium formation by hyphal tip and germ tube. The chs7 mutant also was defective in appressorium penetration and invasive growth. Overall, our results indicate that individual CHS genes play diverse roles in hyphal growth, conidiogenesis, appressorium development, and pathogenesis in M. oryzae, and provided potential new leads in the control of this devastating pathogen by targeting specific chitin synthases.     

Conidial germination and germ tube elongation of Phomopsis amaranthicola and Microsphaeropsis amaranthi on leaf surfaces of seven Amaranthus species: Implications for biological control

Microsphaeropsis amaranthi and Phomopsis amaranthicola are potential biological control agents for several Amaranthus species. In an effort to understand the initial infection processes with these pathogens, a study was conducted of the conidial germination and germ tube length (μm) on the weed leaf surfaces at 21 °C and 28 °C. Weeds included Amaranthus rudis, A. palmeri, A. powellii, A. retroflexus, A. spinosus, A. hybridus, and A. albus. For P. amaranthicola, conidial germination and germ tube length varied among the seven weed species at both temperatures, while for M. amaranthi the differences in germ tube lengths were significant among weed species only at 21 °C. While the conidia of M. amaranthi and P. amaranthicola germinated on the leaf surfaces of all seven weed species, temperature appeared to impact the number and length of germ tubes on the leaf surfaces. The percentage of germinated conidia and the length of germ tubes at both temperatures were often greater for M. amaranthi than for P. amaranthicola. In order for the fungal pathogen to successfully infect and kill a weedy host, conidia must germinate and form a germ tube, two processes that vary with host species and temperature for M. amaranthi and P. amaranthicola. The extent to which successive infection processes, e.g., penetration, invasion and colonization, contribute to host specificity warrants study.