Pathogenicity of four blue-stain fungi associated with aggressive and nonaggressive bark beetles

ABSTRACT The pathogenicity of two isolates of each of four bark beetle-associated blue-stain fungi was evaluated after mass inoculation of about 40-year-old Norway spruce trees (Picea abies). Trees were inoculated with a different isolate of each fungus in 1995 and 1996 at a density of 400 inoculations per m(2) in a 1.2-m-wide band on the lower bole (about 270 inoculations per tree). Trees were felled 15 weeks after inoculation. In 1995, Ceratocystis polonica was the only fungus that had stained the sapwood (56.3% of cross-sectional sapwood area). It induced five times longer phloem necroses, 21 times more dead cambium, and 11 times more dead phloem than any other fungus. In 1996, C. polonica induced less extensive host symptoms and an unidentified Ambrosiella sp. induced comparable symptoms to C. polonica in the phloem and cambium. No trees showed any foliar symptoms 15 weeks after inoculation, but six out of eight trees inoculated with C. polonica in 1995 had only 0 to 25% functional sapwood and probably would have died if felling had been delayed. This study confirms that C. polonica, an associate of the aggressive bark beetle Ips typographus, is pathogenic to Norway spruce. The pathogenicity of the Ambrosiella sp., which is associated with a nonaggressive bark beetle, seems moderate and varies between isolates. The two remaining fungi included in this study (Ophiostoma piceae and a dark fungus with sterile mycelium), which are associated with nonaggressive bark beetles, were nonpathogenic in both experiments. These results are consistent with the hypothesis that aggressive bark beetle species vector virulent fungi that may help them kill trees, but the results also show that some nonaggressive bark beetles may vector phytopathogenic fungi.     

Induction of disease resistance in Norway spruce (Picea abies) by necrotizing fungi

Norway spruce trees ( Picea abies ) preinoculated with the root rot fungus Heterobasidion annosum , Nectria fuckeliana or a pathogenic strain of the blue-stain fungus Ceratocystis polonica were more efficiently protected against a subsequent massive inoculation with pathogenic C. polonica than trees pretreated with nonpathogenic C. polonica or sterile malt agar. Control trees that received no pretreatment were extensively colonized by the mass inoculation. There was a strong negative correlation between the length of the phloem necroses induced by the pretreatment inoculations and the extent of host symptoms caused by mass inoculation with pathogenic C. polonica . The degree of induced resistance in Norway spruce thus depended on the amount of host tissue destroyed by the pretreatment.     

Patterns of bark beetle occurrence in Norway spruce stands of national parks in Tatra Mts. in Poland and Slovakia

This paper presents the results of a research project concerning bark beetle populations in Norway spruce stands managed under a nature protection regime in national parks in Tatra Mountains in Poland and Slovakia. The preliminary data on bark beetle occurrence and related selected stand characteristics are presented. The dynamics of a bark beetle outbreak that occurred from 1994–97 in the stands localized on both sides of the state border and in various nature protection regimes is analyzed retrospectively. The data concerning the tree/stand characteristics, i.e. DBH and increment of trees are discussed in relation to its potential role in the mechanism for the spread of bark beetle populations. The patterns of the spatial distribution of trees attacked during the research period (1999–2001) are described.     

Patterns of bark beetle occurrence in Norway spruce stands of national parks in Tatra Mts. in Poland and Slovakia

This paper presents the results of a research project concerning bark beetle populations in Norway spruce stands managed under a nature protection regime in national parks in Tatra Mountains in Poland and Slovakia. The preliminary data on bark beetle occurrence and related selected stand characteristics are presented. The dynamics of a bark beetle outbreak that occurred from 1994–97 in the stands localized on both sides of the state border and in various nature protection regimes is analyzed retrospectively. The data concerning the tree/stand characteristics, i.e. DBH and increment of trees are discussed in relation to its potential role in the mechanism for the spread of bark beetle populations. The patterns of the spatial distribution of trees attacked during the research period (1999–2001) are described.     

Xylem sap flow of Norway spruce after inoculation with the blue-stain fungus Ceratocystis polonica

In a field experiment, the sap flow of Norway spruce ( Picea abies ) was measured in response to inoculation with Ceratocystis polonica , a virulent fungal associate of the spruce bark beetle Ips typographus. Four vigorous, mature Norway spruce trees were mass-inoculated with C. polonica at a density of four inoculations per dm² within a 120-cm-wide band on the lower bole. Two other trees were inoculated with sterile agar and served as controls. Sap flow of the six trees was measured by the thermal dissipation method from three weeks prior to inoculation until five months after inoculation. In three trees that had been inoculated with the fungus, sap flow density decreased dramatically within the first weeks after inoculation until no sap flow was measurable 4–6 weeks after inoculation. No marked changes in sap flow density were recorded in the fourth tree that had been inoculated with C. polonica , and this tree survived the inoculation. Inoculation with sterile agar did not have any apparent influence on the sap flow of the control trees. The results confirm the ability of C. polonica to kill mature Norway spruce trees. Sap flow measurements by the thermal dissipation method provided a direct and nondestructive method for monitoring the effects of fungal infection on sapwood functionality. Its application for further studies on vascular wilt and vascular stain diseases of trees is therefore recommended.     

Uprooting and shearing resistances in the seedlings of four weedy species

For mechanical weeding, the uprooting force of whole seedlings and the shearing force of basal stems were measured in two grass weeds ( Setaria faberi and Echinochloa crus-galli var. crus-galli ) and two broad-leaved weeds ( Chenopodium album and Amaranthus patulus ), using balance-type equipment and a pair of scissors, respectively. Seedlings were grown in plastic pots. Uprooting and shearing experiments were carried out on the 11th, 16th, 21st, and 26th days after sowing. The uprooting force, the shearing force required to fracture the specimen, plant dry matter weight and cross-sectional area exponentially increased as seedlings grew, but the uprooting stress and shearing toughness maintained similar values after the 2nd sampling time, except for the 3rd and 4th sampling times in C. album . The increases in uprooting force and the shearing force required to fracture the specimen were supported by an expansion of the cross-sectional area of stems of the broad-leaved weeds or pseudostems of grass weeds. The higher values of uprooting stress and shearing toughness in C. album were related to an increased area of lignified organs in the basal stems. There was a significant correlation between uprooting and shearing forces. The shearing force was always higher than the uprooting force, so that most seedlings were uprooted and not fractured at the basal stems or pseudostems. The mean ratio of uprooting force to root dry matter weight after the 2nd sampling time suggested a high uprooting resistance at an extremely low amount of root dry matter weight.     

Using image analysis for quantitative assessment of needle bladder rust disease of Norway spruce

High elevation spruce forests of the European Alps are frequently infected by the needle rust Chrysomyxa rhododendri, a pathogen causing remarkable defoliation, reduced tree growth and limited rejuvenation. Exact quantification of the disease severity on different spatial scales is crucial for monitoring, management and resistance breeding activities. Based on the distinct yellow discolouration of attacked needles, it was investigated whether image analysis of digital photographs can be used to quantify disease severity and to improve phenotyping compared to conventional assessment in terms of time, effort and application range. The developed protocol for preprocessing and analysis of digital RGB images enabled identification of disease symptoms and healthy needle areas on images obtained in ground surveys (total number of analysed images n = 62) and by the use of a semiprofessional quadcopter (n = 13). Obtained disease severities correlated linearly with results obtained by manual counting of healthy and diseased needles for all approaches, including images of individual branches with natural background (R² = 0.87) and with black background (R² = 0.95), juvenile plants (R² = 0.94), and top views and side views of entire tree crowns of adult trees (R² = 0.98 and 0.88, respectively). Results underline that a well‐defined signal related to needle bladder rust symptoms of Norway spruce can be extracted from images recorded by standard digital cameras and using drones. The presented protocol enables precise and time‐efficient quantification of disease symptoms caused by C. rhododendri and provides several advantages compared to conventional assessment by manual counting or visual estimations.     

Infection of Norway spruce (Picea abies (L.) Karst.) seedlings with Pythium irregulare Buism. and Pythium ultimum Trow.: histological and biochemical responses

We have studied the reaction of Picea abies seedlings to infection with Pythium. The highly virulent species Pythium ultimum and the less virulent species Pythium irregulare germinated on the root and hypocotyl surface, formed appressoria and penetrated through the stomata as well as through the epidermis. No major differences in the growth of both fungal species were observed during the early events of colonization. The less virulent species formed about 25% more appressoria suggesting that the fungus experienced difficulties with penetration. Differences were observed in the response of the host plant to infection. Autofluorescence, possibly related to deposition of lignin or lignin-like materials increased more in cortical and endodermal tissue colonized with the highly virulent P. ultimum than with the less virulent P. irregulare. Chitinase activity was highest in the tissues most extensively colonized by the fungus. In addition, a systemic increase of chitinase activity was also detected. Interestingly, chitinase activity increased systemically in cotyledons which were never in contact with the pathogen, indicating the translocation of a systemic signal. Salicylic acid was also detected in spruce seedlings; its level increased in roots during infection with the less virulent P. irregulare.     

4种虫生真菌对烟草甲虫的侵染致死效果

研究了4种虫生真菌对烟草甲虫的致死效果。结果显示,球孢白僵菌对烟草甲虫的防治效果最好,在10⁸个/mL的孢子浓度下,对烟草甲虫的致死中时为10 d。另外几种真菌对烟草甲虫也有不同程度的感染。经过SPSS软件分析了白僵菌对烟草甲虫致死率与时间剂量的量化关系。     

Effect of phenology on susceptibility of Norway spruce (Picea abies) to fungal pathogens

Ceratocystis polonica and Heterobasidion parviporum are important fungal pathogens in Norway spruce (Picea abies). Tree susceptibility to these pathogens with respect to phenology was studied using artificial fungal inoculations at six stages of bud development, and assessed by measuring phloem necroses in the stems of 2‐ and 8‐year‐old trees. Tree capacity for resistance was assessed by measuring phloem nonstructural carbohydrates at each stage. Phloem necroses were significantly larger in trees with fungal versus control inoculations and increased significantly over time. Changes in nonstructural carbohydrates occurred in the trees; a significant decline in starch and a slight but significant increase in total sugars occurred over time. These results suggest that susceptibility to fungal pathogens and carbohydrate levels in the stems of the trees were related to fine‐scale changes in bud development. A trade‐off may occur between allocation of starch (the major fraction of the stem carbohydrate pool) to bud development/shoot growth versus defence of the stem. Previous tests of plant defence hypotheses have focused on herbivory on plants growing under different environmental conditions, but the role of phenology and the effect of pathogens are also important to consider in understanding plant resource allocation patterns.     

小麦全蚀病菌不同致病力菌株的致病特点

利用小麦种子根接种小麦全蚀病菌Gaeumannomyces graminis var．tritici，研究了不同致病力菌株的致病特点。结果表明，弱致病菌株可侵染小麦，但罹病过程缓慢，接种第5天仅在皮层观察到少量菌丝体，13天有少量菌丝进入中柱，中柱组织在菌丝侵入前褐变，未出现导管堵塞现象，也不能导致典型的黑根症状。强致病菌株接种第2天可侵入皮层，8天即进入中柱，并在寄主组织内产生大量菌丝体，致使寄主皮层组织和中柱细胞大量褐变和坏死，以及导管堵塞。     

白龙江林区云杉苗圃地下害虫及天敌昆虫群落结构

应用多样性指数、均匀度指数、优势度和群落相似性指数对白龙江林区内不同生境下的云杉苗圃地下害虫及天敌昆虫的群落结构进行了研究。结果表明,3种生境下的云杉苗圃地下害虫优势种群明显,迭部安子沟苗圃以小云斑鳃金电、云杉树叶象、粗绿彩丽金龟为优势种;舟曲沙滩苗圃、白水江中路河苗圃以小云斑鳃金龟、云杉树叶象、蓝边矛丽金龟、大云斑鳃金龟为优势种。主要天敌有中华疱步甲、步甲。休闲1年后的苗圃地多样性指数、均匀度相对较大。     

内生真菌对青贮玉米幼苗抗旱性的影响

为了研究干旱胁迫下内生真菌对青贮玉米幼苗生长的影响并筛选可以提高青贮玉米幼苗抗旱性的菌株,开展盆栽试验,将分离自西鄂尔多斯荒漠强旱生孑遗植物四合木、沙冬青、绵刺等植物根系的8株内生真菌和1株内生真菌模式种印度梨形孢,以灌根的方式分别接种到青贮玉米幼苗根系,镜检侵染率后,采用PEG6000模拟重度干旱胁迫培养2周,测定青贮玉米幼苗生理生化指标并计算抗旱系数,利用主成分分析法综合评价内生真菌对青贮玉米幼苗抗旱性的影响。结果表明：所有菌株均能侵入青贮玉米根系形成共生,但侵染率存在差异;在未接菌（E-）组的非胁迫和胁迫植株之间,地上部分鲜重、叶相对含水量、叶绿素含量、根体积、根表面积、根鲜重、根干重、根干物质含量、Pro含量、MDA含量、CAT活性、SOD活性、POD活性等13个指标存在显著差异,基于13个生理指标的主成分分析表明,只有Am04、Tm36、Tm02、印度梨形孢等4株内生真菌可提高青贮玉米幼苗抗旱性,并以菌株Am04最优;而Pm53、Tm30、Pm38、Pm06、Pm57等5株内生真菌并未提高青贮玉米幼苗抗旱性;且侵染率和大多数抗旱性生理指标之间无相关性。因此,不同内生真菌对青贮玉米幼苗抗旱性具有不同程度影响,其中菌株Am04较其他菌株更有利于提高青贮玉米幼苗抗旱能力。     

Effect of temperature on virulence of Rhizoctonia solani on soybean leaves and seedlings

Isolates representing 11 anastomosis groups (AGs) of Rhizoctonia solani from various geographic locations and host plants were tested for virulence on soybean leaves at 15, 20, 25, 30, and 35°C, and on soybean seedlings at 20, 25, and 30°C. Numbers of infection cushions formed on soybean leaves were determined using light microscopy. Isolates of AG-1 IA, AG-1 IB and AG-5 were more virulent on soybean leaves at 20, 25, and 30°C than isolates of AG-1 IC and AG-4. Maximum numbers of infection cushions were formed on soybean leaves by AG-1 (IA, IB, and IC), AG-4, and AG-5 at 25 and 30°C. The other AGs tested did not form infection cushions on soybean leaves although some caused minimal disease severity. Isolates of AG-1 IA formed significantly more infection cushions and caused greater disease severity than AG-1 IB and other isolates at 35°C. Maximum seedling infection, based on per cent area of hypocotyl region covered by lesions occurred at 25 C for AG-1 (IA, IB, and IC) and AG-4. Isolates of AG-5 caused greater seedling infection at 20°C than at 25 and 30°C. The other AGs tested caused only minimal damage to the seedlings. Isolates of AG-4 and AG-5 are not known to cause Rhizoctonia foliar blights of soybean in Louisiana, but their potential to be destructive foliar pathogens is confirmed.     

雷帕霉素对4种植物病原真菌的抑菌活性

采用菌丝生长速率法,测定了雷帕霉素对番茄灰霉病菌、油菜菌核病菌、水稻纹枯病菌和棉花枯萎病菌的抑菌活性,比较了嘧菌酯、丙烷脒及雷帕霉素对番茄灰霉病菌的抑菌效果,并通过电子显微镜观察了雷帕霉素对番茄灰霉病菌菌丝生长的影响。结果表明:雷帕霉素对供试4种植物病原真菌菌丝均表现出了极强的抑制活性,其中对油菜菌核病菌的抑制作用最强,EC50值为2.23×10-4μg/mL,对番茄灰霉病菌、水稻纹枯病菌和棉花枯萎病菌的EC50值分别为1.32×10-3、4.05×10-3及3.82×10-3μg/mL;雷帕霉素对番茄灰霉病菌菌丝的抑制活性显著高于嘧菌酯(EC50值为3.24μg/mL)和丙烷脒(EC50值为3.81μg/mL)。电镜观察发现,经雷帕霉素处理后,番茄灰霉病菌菌丝表现出提前衰老等症状。研究结果可为深入探讨雷帕霉素对植物病原真菌的作用机制奠定基础。     

稻田土壤8种真菌对4种杀菌剂的敏感性分析

基于ITS-5.8s rDNA区序列,初步鉴定了浙江大学试验基地稻田土壤的8种常见真菌。采用菌丝生长速率法测定了井冈霉素、多菌灵、咪鲜胺和三唑酮4种常用杀菌剂对这8种土壤真菌的抑菌活性,以及多菌灵与咪鲜胺混用对禾柄锈菌、黑曲霉和长枝木霉3种真菌菌丝生长的抑制作用。结果表明:8种真菌对咪鲜胺最敏感,EC50值在0.091~3.1 mg/L之间;多菌灵的EC50值在0.15~1.8 mg/L之间;三唑酮的EC50值在0.57~8.5 mg/L之间;井冈霉素的敏感性最低,EC50在12~94 mg/L之间。由4种杀菌剂的SSDs曲线分析结果得出,不同真菌对杀菌剂敏感性存在显著差异。当多菌灵与咪鲜胺按其EC50值分别以体积比3∶2、1∶1和7∶3混用时,增效系数分别为1.615、1.039和1.042,表现出一定的协同或相加作用。本研究结果为进一步阐明杀菌剂对稻田生态系统真菌群落的生态效应及杀菌剂的科学合理使用提供了重要依据。     

噻霉酮和苯醚甲环唑混配对4种不同病原菌的增效作用

采用室内生长速率法,以黄瓜菌核病菌、棉花立枯病菌、番茄早疫病菌和辣椒炭疽病菌为作用目标,研究了噻霉酮和苯醚甲环唑混配对病菌的增效作用。苯醚甲环唑对4种病菌的EC50为0.650 9～1.211 2mg/L,噻霉酮对4种病菌的EC50为20.887 0～43.514 6mg/L。当苯醚甲环唑和噻霉酮的混配比例为1∶10时,对黄瓜菌核病菌的共毒系数为519.11;混配比例为1∶2时,对辣椒炭疽病菌的共毒系数为230.66;混配比例为1∶5时,对棉花立枯病菌和番茄早疫病菌的共毒系数分别为219.42和190.71。噻霉酮和苯醚甲环唑按适当比例混配对4种不同病原菌具有明显的增效作用。     

群体感应系统对甜瓜果斑病菌MH21致病力的影响

 以N-乙酰高丝氨酸内酯（N-acyl-homoserine lactone, AHL）为信号分子的群体感应（quorum-sensing, QS）系统是很多病原细菌的重要致病性调控因子。本文自甜瓜果斑病菌——西瓜食酸菌（Acidovorax citrulli）菌株MH21中克隆到AHL信号合成基因luxI_MH21,并构建了其缺失突变体MΔluxI_MH21及转化有AHL信号降解酶编码基因aiiA和aidH的工程菌株MAiiA和MAidH。信号检测结果显示MΔluxI_MH21、MAiiA和MAidH菌株均无AHL信号产生,同时细菌的游动能力及在基本培养基中的生长速率均显著下降,但对细菌生物膜形成和在非寄主植物烟草上诱导过敏性坏死反应的能力没有影响。盆栽条件下,经低浓度（10⁴ CFU/mL）MΔluxI_MH21、MAiiA和MAidH菌株处理的甜瓜种子萌发后幼苗死亡率显著低于野生型MH21和luxI_MH21基因互补菌株MΔluxI_MH21HB的处理;而高浓度细菌（10⁸ CFU/mL）处理种子后,除MAidH菌株处理引起的死苗率明显低于野生型MH21处理,其他菌株与MH21没有显著差异。子叶注射试验也得到相似的结果,以低浓度细菌（10⁴ CFU/mL）注射甜瓜子叶后发现MΔluxI_MH21、 MAiiA和MAidH菌株甜瓜子叶中的繁殖速率及对子叶的致病力与野生型MH21相比均显著下降;而高浓度细菌（10⁸ CFU/mL）处理子叶时,MΔluxI_MH21和MAiiA菌株与野生菌MH21相比致病力无显著差异,仅有MAidH菌株的致病力明显下降。说明QS系统影响菌株MH21在低细菌浓度下对甜瓜幼苗的致病力,这种作用可能与影响细菌生长有关。