真菌病毒减弱寄主致病性机理及其生防应用

真菌病毒是在真菌体内复制和增殖的病毒,在真菌中广泛存在。农业生产中70%~80%的植物病害由病原真菌侵染导致,真菌病毒能够减弱植物病原真菌的致病性,还可以将其转换为内生真菌从而促进植物生长和抗病,因此真菌病毒是一类重要的生防资源。本文综述了真菌病毒减弱寄主致病性机理及其在病害防治中的应用,同时提出了真菌病毒用于生物防治的前景和面临的问题,为植物真菌病害的生物防治提供参考。     

Plasticity in plant-microbe interactions: a perspective based on the pitch canker pathosystem

What we know about the life history of fungi that cause disease in plants is commonly based on studies of the pathogen’s interaction with a susceptible host: how and when infection occurs, growth and reproduction within the host, and survival during the interval when a growing host is not available. This focus is appropriate, given the need for information that will facilitate management of disease affecting an economically important crop, but it can limit recognition of the full range of resources that may be utilized by fungi that we classify as plant pathogens. This was certainly the case for Fusarium circinatum, which causes a destructive disease of pines known as pitch canker. Although F. circinatum was initially known only as a necrotrophic, wound-infecting pathogen of coniferous trees, recent research has revealed that an isolate of this fungus that will kill shoot tissue when inoculated into a wound can also have a biotrophic relationship with roots of pine seedlings, infect and grow within grasses without causing symptoms, and cause ear rot of corn. Thus, although F. circinatum became known to science because it induced visible symptoms on pines, it has the capacity for a much broader range of ecological activities than is captured by its designation as a necrotrophic pathogen. The physiological plasticity manifested by F. circinatum illustrates the challenge of obtaining a comprehensive understanding of the life history of a plant pathogenic fungus.     

Resistance assessment and biochemical responses of sugar beet lines against <Emphasis Type="Italic"> Pythium aphanidermatum,</Emphasis> causing root rot

Wood-staining fungi, moulds and decay fungi colonize freshly cut wood. Of these, only ophiostomatoid fungi are considered major agents of sapstain on logs and freshly sawn lumber because they can cause permanent staining and discoloration. Sapstain in pine pallets significantly reduces their market value and use in the food industry. The aim of this study was to identify the sapstaining fungi that colonize Scots pine wood used in pallet production. In addition, we evaluated the growth and stain intensity of six isolates of ophiostomatoid fungi on freshly cut Scots pine billets. Fungi were isolated from samples of Scots pine and identified based on morphology and DNA sequence comparisons for three gene regions (ITS, β-tubulin, TEF-1α). A total of 1259 isolates representing 31 fungal species were obtained from the pine samples in Poland during July and September. The isolates represented different ecological and taxonomical groups and belonged to the categories of staining fungi, decay fungi and surface moulds. The most frequently isolated fungi were ophiostomatoid fungi (14 species including an unknown Leptographium sp. and Ophiostoma sp.) and moulds (mainly Trichoderma and Mucor spp.). Of the ophiostomatoid species, Ophiostoma floccosum, O. piceae, O. piliferum and Endoconidiophora pinicola were the predominant species. The results of pine billet inoculation showed that among the six fungal species tested, E. pinicola exhibited the fastest growth in all three directions on the billets. Ophiostoma minus and O. piliferum displayed moderate growth rates in pine billets, while O. floccosum, O. piceae, and Leptographium lundbergii grew very slowly, especially in the tangential and radial directions. The information provided in this paper will help develop more effective control strategies for sapstain prevention in Scots pine.     

Genetic diversity and identification of race 3 of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lactucae</Emphasis> in Taiwan

Sorghum is an important drought tolerant crop cultivated for food and fodder purposes. Anthracnose disease caused by Colletotrichum graminicola is a major constraint in sorghum productivity in India. Certain antagonistic fungi, that were isolated in the previous study from the rhizosphere and rhizoplane of perennial grasses in India, were studied for their antagonism in vitro to C. graminicola, root colonization ability and rhizosphere competence. Out of 138 isolates tested, 89 were antagonistic. Fifteen fungal isolates with greater than 70 % in vitro inhibition zone to the pathogen tested positive for root and rhizosphere colonization abilities. Three isolates – Chaetomium globosum isolate 57, Trichoderma harzianum isolate 184 and Fusarium oxysporum (NSF isolate 9) with prominent biocontrol potentials were tested for the control of sorghum anthracnose in greenhouse and field. Chaetomium globosum, Trichoderma harzianum and Fusarium oxysporum isolates decreased seedling mortality, and incidence and severity of disease at different growing stages. They promoted plant growth (dry biomass- 45.3, 40.0 and 46.7 %) and increased yield (grain biomass- 33.3, 23.8 and 49.2 %) respectively, over control in field. The population of the above fungi in soil was moderately high at harvest stage. The present investigation revealed that fungal isolates from rhizosphere and rhizoplane of perennial grasses could be employed to manage anthracnose and enhance plant growth and yield potentialities in sorghum, at the same time.     

Characterization of three Colletotrichum acutatum isolates from Capsicum spp.

Colletotrichum acutatum causes anthracnose on peppers (Capsicum spp.), resulting in severe yield losses in Taiwan. Fungal isolates Coll-153, Coll-365 and Coll-524 collected from diseased peppers were found to differ in pathogenicity. Pathogenicity assays on various index plants revealed that Coll-524 was highly virulent and Coll-153 was moderately virulent to three commercially available pepper cultivars. Both isolates induced anthracnose lesions and produced abundant conidia. Coll-365 was only weakly virulent on pepper fruit, where it caused small lesions and hardly produced conidia on pepper fruit. However, Coll-365 was highly pathogenic to tomato fruit and mango leaves, where it caused anthracnose lesions and formed acervuli and conidia. All three isolates showed similar abilities in the attachment and germination of conidia, formation of highly branched hyphae and appressoria, penetration of cuticles, and infection of epidermal cells on chili peppers. Coll-365 accumulated less turgor pressure in appressoria but produced higher levels of cutinase and protease activity than Coll-153 and Coll-524 did. All three isolates invaded the neighbouring cells through plasmodesmata in chili peppers and showed similar pectinase or cellulase activities in culture. However, the most virulent strain Coll-524 expressed stronger laccase activity and was more resistant to capsaicin compared to Coll-153 and Coll-365. The three isolates are different in numbers and sizes of double-stranded RNAs. Depending on the cultivar genotypes, cellular resistance of chili pepper to C. acutatum might rely on the ability to restrict penetration, colonization, or conidiation of the pathogen. We conclude that the differences in pathogenicity among the three C. acutatum isolates of pepper are attributed to their ability to colonize the host plant.     

Characterization of phenotypic variants of Clavibacter michiganensis subsp. michiganensis isolated from Capsicum annuum

Phenotypic variants of Clavibacter michiganensis subsp. michiganensis (Cmm) were isolated from pepper fields and from pepper seeds during quarantine inspections. All strains isolated from pepper (pepper isolates) produced orange-coloured colonies with lower mucoidy than typical Cmm strains isolated from tomato (tomato isolates). However, the results of ELISA, fatty acid analysis, 16S rDNA sequencing, and PCR analysis showed that all pepper isolates were similar enough to be identified as Cmm. In addition to phenotypic variations, the pepper isolates showed different pathogenic and genetic characteristics from tomato isolates from the USA, Europe, or other countries. They could be clearly distinguished in terms of pathogenicity, as they showed increased pathogenicity to pepper but reduced pathogenicity to tomato. Tomato isolates caused strong wilting and canker in tomato, but caused only canker and no wilting in pepper and bell pepper. However, pepper isolates caused no wilting, even in tomato, and only caused canker in the three host plants. In addition, compared to tomato isolates, pepper isolates showed increased colonization efficiency and caused a greater reduction in shoot dry weight in pepper. Pepper and tomato isolates could be separated into two groups according to host origin on the basis of 16S rDNA and ITS sequence analysis. They also showed different rep-PCR genomic fingerprints. All pepper isolates showed higher cellulase activity than tomato isolates on M9CMC plates. However, two plasmid-borne virulence genes of Cmm, pat-1, and celA, were not detected in any pepper isolates by PCR. Furthermore, PCR for pathogenicity-related genes located on a pathogenicity island (PAI) revealed that all tomato isolates were positive for these genes, whereas the pepper isolates did not show any PCR products for the chpC, chpG, ppaA, or tomA genes. Therefore, we suggest that the pepper isolates may represent a separate Cmm population that has evolved within the limits of this host.     

Infection by Magnaporthe oryzae chrysovirus 1 strain A triggers reduced virulence and pathogenic race conversion of its host fungus, <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Magnaporthe oryzae chrysovirus 1 strain A (MoCV1-A) is associated with an impaired growth phenotype of its host fungus, Magnaporthe oryzae. In this report, we assayed the virulence and pathogenicity of MoCV1-A-infected and MoCV1-A-free M. oryzae on rice plants. MoCV1-A infection did not affect virulence-associated fungal traits, such as conidial germination and appressorium formation. However, after punch inoculation of leaves on rice plants, MoCV1-A-infected strain formed smaller lesions than the MoCV1-A-free strain did on all rice varieties tested, showing that MoCV1-A infection resulted in reduced virulence of host fungi in rice plants. In contrast, after spray inoculation of rice seedlings, in some cases, MoCV1-A-infected and MoCV1-A-free strains caused different lesion types (resistance to susceptible, or vice versa) on individual international differential rice varieties. However, we did not find any gain/loss of the fungal avirulence genes by PCR, suggesting that MoCV1-A infection can convert the pathogenicity of the host M. oryzae from avirulence to virulence, or from virulence to avirulence, depending on the rice variety. We also confirmed the correlation of these race conversion events and invasive hyphae growth of the fungi in a leaf sheath inoculation assay. These data suggested that MoCV1-A infection generally confers hypovirulence to the fungal host and could be a driving force to generate physiological diversity, including pathogenic races.     

Cyanobacteria mediated plant growth promotion and bioprotection against Fusarium wilt in tomato

Cyanobacteria - phytopathogenic fungi - tomato plant interactions were evaluated for developing suitable biological options for combating biotic stress (Fusarium wilt) and enhancing plant vigour. Preliminary evaluation was undertaken on the fungicidal and hydrolytic enzyme activity of the cyanobacterial strains (Anabaena variabilis RPAN59, A. laxa RPAN8) under optimized environmental/nutritional conditions, followed by amendment in compost-vermiculite. Such formulations were tested against Fusarium wilt challenged tomato plants, and the Anabaena spp. (RPAN59/8) amended composts significantly reduced mortality in fungi challenged treatments, besides fungal load in soil. Cyanobacteria amended composts also led to an enhancement in soil organic C, nitrogen fixation, besides significant improvement in growth, yield, fruit quality parameters, N, P and Zn content. The tripartite interactions also enhanced the activity of defence and pathogenesis related enzymes in tomato plants. A positive correlation (r?=?0.729 to 0.828) between P content and pathogenesis/defense enzyme activity revealed their role in enhancing the resistance of the plant through improved nutrient uptake. Light and scanning electron microscopy (SEM) revealed cyanobacterial colonization, which positively correlated with reduced fungal populations. The reduced disease severity coupled with improved plant growth/ yields, elicited by cyanobacterial treatments, illustrated the utility of such novel formulations in integrated pest and nutrient management strategies for Fusarium wilt challenged tomato crop.     

Use of Fusarium graminearum transformed with gfp to follow infection patterns in barley and Arabidopsis

The fungal pathogen Fusarium graminearum attacks the seed spikes of barley and wheat, causing sterility, reduced seed weight and accumulation of mycotoxins. To explore infection patterns in barley and in the Arabidopsis model system, the green fluorescent protein gene (gfp) was used to transform F. graminearum. Inoculation of intact barley spikes resulted in rapid colonization of the brush hairs (ovary epithelial hairs) at the extruded seed tip within 7 h. Colonization followed a pattern of rapid basipetal growth along the pericarp epithelium (interior to the lemma and palea), accompanied by slower growth inward through the pericarp and testa. However, at 16 days after infection the aleurone and starchy endosperm remained uninfected, despite heavy colonization of the pericarp. Colonization of the outer lemma also occurred but was much slower. No increase in amylase enzyme activities was found, discounting the possibility that F. graminearum utilizes gibberellin-induced host enzymes to tap the endosperm for nutrients. The transformed Fusarium strain readily infected Arabidopsis thaliana leaves and produced copious spores within distant leaves. Results show the utility of gfp in tracing the growth of this pathogen, without misinterpretation due to contaminating fungi, and for resistance studies utilizing the Arabidopsis model system.     

Nursery‐linked plantation outbreaks and evidence for multiple introductions of the pitch canker pathogen Fusarium circinatum into South Africa

In recent years, Pinus plantation forestry has been significantly hampered by outbreaks of pitch canker caused by the fungus Fusarium circinatum. This study investigated the role of Pinus host, geographic origin and reproductive mode in structuring the F. circinatum populations in plantations. For this purpose, 159 isolates originating from diseased plantation trees in the Western and Eastern Cape Provinces of South Africa were genotyped using 10 microsatellite markers. Analyses of these data revealed 30 multilocus haplotypes and that the populations were distinct based on geographic origin as well as host. However, shared haplotypes were observed between populations, showing that these populations are connected, possibly through the movement of haplotypes. A second aim was to determine whether the genetic variation found in these populations of the fungus could be attributed to outbreaks of the seedling disease caused by this pathogen in Pinus nurseries. To achieve this goal, an additional set of 43 isolates originating from pine seedling nurseries was genotyped and analysed. The results showed that the populations of F. circinatum in plantations most probably originated from the nursery outbreaks that occurred prior to the plantation outbreak. Inferences regarding reproductive mode further showed that sexual reproduction has little impact on the genetic makeup of the F. circinatum populations and that they primarily reproduce asexually. Overall, the results of this study showed that the F. circinatum diversity in South Africa has arisen due to multiple introductions of the pathogen and is not due to sexual reproduction.     

New insights into radiata pine seedling root infection by Fusarium circinatum

Pine root infection by Fusarium circinatum has been reported in the literature, but the underlying pathogenic interaction is poorly understood. A green fluorescent protein (GFP)‐tagged F. circinatum isolate, together with confocal microscopy, was used in order to monitor the events associated with root infection of Pinus radiata seedlings. It was found that in order to reach and successfully infect pine roots, F. circinatum employed features that are similar to those previously described for other root‐infecting pathogens, such as mycelial strands, single runner hyphae and simple hyphopodia as well as other features that are reminiscent of those that are known to be involved in biotrophic invasion, such as bulbous invasive hyphae and filamentous invasive hyphae. Abundant sporulation was observed at the root surface as well as inside tracheids both in roots and in the root collar region. The fungus can spread from the roots to the aerial parts of the plant, and once there, colonization appears to be similar to the process that occurs when the pathogen is inoculated in the stem. Wilting symptoms and plant demise may be the result of a reduction in water uptake by roots and of the blockage of the vascular system by fungal hyphae and resin.     

Mapping of easy to screen SSR markers for selection of RFLP markers-bracketed downy mildew resistance QTLs in pearl millet

There is a growing necessity to replace chemical agents with ecofriendly materials, arising from the impact on the environment and/or human health, which calls for the design of new broad-spectrum fungicides. In this work, chitosan oligomers (COs), propolis (Ps) and silver nanoparticles (AgNPs) mixtures in solution were assessed to control the growth of different phytopathogenic fungi and oomycetes in vitro. Binary solutions of COs-Ps and COs-AgNPs evinced the highest antifungal effect against Fusarium circinatum and Diplodia pinea fungi, respectively, with a ca. 80% reduction in their mycelial growth. The COs solution by itself also proved to be greatly effective against Gremmeniella abietina, Cryphonectria parasitica and Heterobasidion annosum fungi, causing a reduction of 78%, 86% and 93% in their growth rate, respectively. Likewise, COs also attained a 100% growth inhibition on the oomycete Phytophthora cambivora. On the other hand, Ps inhibited totally the growth of Phytophthora ×alni and Phytophthora plurivora. The application of AgNPs reduced the mycelial growth of F. circinatum and D. pinea. However, the AgNPs in some binary and ternary mixtures had a counter-productive effect on the anti-fungal/oomycete activity. In spite of the fact that the anti-fungal/oomycete activity of the different treatments showed a dependence on the particular type of microorganism, these solutions based on natural compounds can be deemed as a promising tool for control of tree diseases.     

A laboratory bioassay for evaluating pathogenicity of Macrophomina phaseolina and Rhizoctonia solani isolates to strawberry stolons

Macrophomina phaseolina and Rhizoctonia spp. are the most important soilborne pathogens of the strawberry crop that cause seriously reduced yields. Various methods are being used to determine pathogenicity of these fungi isolated from strawberry; however, they take a long time to grow strawberry plants. They also encounter some problems to provide a large number of healthy plants for the pathogenicity tests. Therefore, the aim of this study was to develop a faster and more reliable pathogenicity test for soilborne fungal pathogens of strawberry by using the stolons of healthy strawberry plants. The stolon inoculation method was demonstrated to be easy and rapid for properly distinguishing soilborne fungal species of strawberry, whether pathogenic or non-pathogenic, and also for assessing the isolates for virulence.     

Unique clones of the pitch canker fungus, <Emphasis Type="Italic">Fusarium circinatum,</Emphasis> associated with a new disease outbreak in South Africa

Pitch canker of pines is caused by the fungus Fusarium circinatum. In South Africa, this pathogen has mostly been a nursery problem. From 2005, however, outbreaks of pitch canker have been reported from established Pinus radiata and P. greggii in the Western and Eastern Cape Provinces. Most recently, pitch canker-like symptoms were observed on 10-year-old P. greggii trees in a plantation in the midlands of the KwaZulu-Natal (KZN) Province. The aim of this study was to: (i) identify the causal agent of the observed symptoms, (ii) determine the genetic diversity, and (iii) the mode of reproduction of this fungal population. Furthermore, the aggressiveness of isolates from these trees was compared with that of isolates obtained previously from P. patula in South Africa. Isolates from the P. greggii trees in KZN were confirmed as F. circinatum based on both morphology and DNA sequence analyses. Microsatellite marker analyses revealed the presence of five genotypes of F. circinatum, not previously reported from other plantations in South Africa, with one of these genotypes being dominant. These genotypes were all pathogenic to P. patula and P. elliottii. No evidence of sexual reproduction was detected in the KZN population of the fungus. This was consistent with the fact that isolates from P. greggii were all of the MAT-2 mating type, in contrast to previously collected isolates from across South Africa that included both mating types. The results suggest that the outbreak of pitch canker on P. greggii in KZN represents a separate introduction of F. circinatum into the region with important implications for managing the disease.     

Cell wall-degrading enzymes of Didymella bryoniae in relation to fungal growth and virulence in cantaloupe fruit

Didymella bryoniae is an important pathogen of cucurbits worldwide. Virulence factors of D. bryoniae were investigated in regard to fungal growth and the production of the cell wall-degrading enzymes, polygalacturonase (PG), pectate lyase (PL), pectin lyase (PNL), β-galactosidase (β-Gal) and cellulase (Cx). Virulence levels of five D. bryoniae isolates were determined by the severity of inoculated cantaloupe fruit decay. The highly virulent isolates had more mycelial growth than the moderately virulent isolates in different media. PG activities produced by the highly virulent isolates in shake cultures and in decayed fruit were greater than those of the moderately virulent isolates. PNL, but not PL, in decayed fruit was higher with the highly virulent isolates compared to the moderately virulent ones. The highly virulent isolates showed higher Cx activity than the moderately virulent ones in decayed fruit and in fruit tissue shake culture. β-Gal activities of the highly virulent isolates in pectin shake culture and in decayed fruit were greater than those of the two moderately virulent isolates although fruit also produced β-Gal. Protein analysis showed two fungal β-Gal isozymes in decayed fruit compared to those of healthy fruit. Correlation analysis indicated that the activities of PG, PNL, β-Gal and Cx in cultures and in decayed fruit positively correlated with fungal growth and fruit decay severity. The results of this study suggest that PG, PNL, β-Gal, and Cx appear to be virulence factors of D. bryoniae in cantaloupe decay with PG and β-Gal as the most predominant fruit decay enzymes.     

来源于湖北省梨轮纹病菌中真菌病毒多样性检测及其分析

 一些真菌病毒严重影响寄主生长、产孢量、致病力。本研究对来源于湖北梨轮纹病菌携带真菌病毒情况进行分析,旨在挖掘具有生防潜能的真菌病毒资源。随机采集湖北省梨树发病枝干,分离、鉴定获得13个梨轮纹病菌株,其生长速率、致病性存在差异。提取dsRNA检测,发现12个菌株均携带不同大小的dsRNA片段。观察其dsRNA带型,HBWH-14 dsRNA含有1~7 kb 9条带,其他菌株携带的dsRNA条带位置均位于HBWH-14所含dsRNA带型中的某1条或多条对应的条带位置。RT-PCR检测发现部分菌株携带产黄青霉病毒BdCV1、双分体病毒BdPV1、单分体病毒BdTV1以及未分类的BdRV1。本研究未发现梨轮纹菌株所含病毒种类与致病力存在直接的相关性。HBWH-14原生质体脱毒获得后代100%携带的dsRNAs稳定遗传。     

Correlation of in planta endo‐beta‐1,4‐xylanase activity with the necrotrophic phase of the hemibiotrophic fungus Mycosphaerella graminicola

The association of the cell wall degrading enzyme endo‐beta‐1,4‐xylanase (EC 3.2.1.8) with pathogenicity of Mycosphaerella graminicola was examined in planta. The enzyme production of two M. graminicola isolates (T0372 and T0491), as well as their ability to infect seedlings of susceptible wheat cv. Scorpion, was first compared. No significant difference was found between the two isolates regarding spore germination rates, mycelial growth on the leaf surface or direct and stomatal penetrations. However, restricted hyphal growth was observed inside leaves inoculated with T0372, whereas successful mesophyll colonization with a strong intercellular fungal growth was found in leaves infected with T0491. Likewise, T0372 was unable to induce lesions bearing pycnidia and to produce endo‐beta‐1,4‐xylanase activity until 22 days post‐inoculation (d.p.i.). On the other hand, significant high increases of both diseased leaf area bearing pycnidia and endo‐beta‐1,4‐xylanase activity were observed between 16 and 22 d.p.i. for T0491 (r = 0·98). The investigation of 24 additional isolates, including the IPO323 and IPO94269 reference isolates, highlighted a strong correlation between endo‐beta‐1,4‐xylanase activity and disease development levels (r = 0·94). This study demonstrates that differences in pathogenicity in M. graminicola are not linked to events on the leaf surface or to frequency of leaf penetration, but to the ability of the fungus to colonize the mesophyll and to produce the cell wall degrading enzyme endo‐1,4‐beta‐xylanase during the necrotrophic phase.     

五株病原真菌对桃小食心虫的致病力

为筛选桃小食心虫高致病性菌株,以在山西省自然感病的桃小食心虫越冬幼虫上分离获得的5株病原真菌为材料,研究各菌株感染桃小食心虫幼虫的症状、对寄主的致死率,以及与菌株毒力相关的胞外类枯草杆菌蛋白酶、几丁质酶和脂肪酶的活性变化,并评价其生物防治潜力。5个菌株在寄主上的感病症状和致死率有显著差异,感病后10天,球孢白僵菌BbTST05对寄主的致死率最高,为89.3%,其类枯草杆菌蛋白酶、几丁质酶和脂肪酶3种酶活性均最大;粉棒束孢IfTSL02对寄主的致死率及其蛋白酶、几丁质酶活性次之;尖孢镰孢菌的FoTSL01、FoTSL03和FoTSL04菌株对桃小食心虫的致死率均低于61%,3种胞外酶的活性也较低。因此,球孢白僵菌BbTST05和粉棒束孢IfTSL02可用于桃小食心虫的防治。