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1.
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2.
为揭示向日葵大丽轮枝菌Verticillium dahliae Kleb.的致病机理,利用农杆菌介导法将带有潮霉素抗性标记和绿色荧光蛋白报告基因的双元载体转入大丽轮枝菌的分生孢子中并获得阳性转化子,以野生型菌株为对照,对随机挑取的阳性转化子的菌落形态、菌丝生长速率、产孢量、粗毒素分泌量和致病力进行了研究。结果表明,共获得800株阳性转化子,随机选取的40株阳性转化子中有2株的菌落只产生白色气生菌丝,不能形成黑色微菌核。与对照相比,40株转化子的生长速率均有不同程度降低,其中转化子A1生长速度降低最显著,菌落直径仅为3.28 cm,比对照下降了38.92%。40株转化子中有3株的产孢量高于对照,其中转化子A9的产孢量最高,为3.50×10~7个/mL,比对照提高1.10倍;转化子A1的产孢量最低,仅为1.35×10~7个/mL,比对照下降了19.16%。40株转化子中有4株的粗毒素分泌量较对照显著升高,占测定菌株的10%,有24株较对照显著降低,占60%,其余12株与对照无显著差异。40株转化子中有3株的致病力较对照显著增强,占测定菌株的7.5%;有7株的致病力较对照显著降低,占17.5%;其余30株与对照无显著差异。  相似文献   

3.
Liu Q  Xiao CL 《Phytopathology》2005,95(5):572-580
ABSTRACT Potebniamyces pyri is the causal agent of Phacidiopycnis rot, a postharvest disease of pears. Infection of fruit occurs in the orchard, and symptoms develop during storage. Conidial germination of P. pyri in response to nutrient, temperature, wetness duration, relative humidity (RH), and pH was determined in vitro. Conidia germinated by either budding or developing germ tubes in various concentrations of pear juice solutions. The mode of conidial germination was nutrient-dependent. Low nutrient levels favored budding, whereas high nutrient levels favored germ tube development. Conidia germinated at 0 to 30 degrees C but not at 35 degrees C, with optimum temperature between 20 and 25 degrees C. Wetness durations of 4 to 5 h and 6 to 8 h at optimum temperature were required for budding and developing germ tubes, respectively, and 20 to 24 h of wetness was required to reach germination peaks. Regardless of temperature, conidia germinated primarily by budding in 10% pear juice. Secondary conidia, produced by budding of conidia, initially increased their dimensions and later germinated at 0 to 25 degrees C in the same manner as mother conidia. No germination of secondary conidia occurred at 30 degrees C. Germ tubes from conidia elongated at 0 to 25 degrees C but not at 30 degrees C. No germination occurred at 相似文献   

4.
This fluorescence and immunoelectron microscopic study showed that β-1,3-D-glucan accumulated only in leaves of a susceptible cultivar of Japanese pear after treatment with a host-specific toxin, AK-toxin I, from Alternate, alternata Japanese pear pathotype. The positive fluorescent reaction of callose was detected only in aniline blue fluorochrome-stained sections from toxin-treated leaves of the susceptible cultivar: positive sites were observed on cell walls of leaf cells. The sites of callose deposition were probably consistent spatially with modified sites on the plasma membrane that were observed only in the toxin-treated leaves of the susceptible cultivar. The toxin-induced modifications, identified as damage to the plasma membrane, were characterized by invagination of the plasmalemma specifically at plasmodesmata and as the concomitant accumulation of extracellular polysaccharides at the invaginated sites. A positive reaction to anti-β-1,3-D-glucan antibody was detected at the polysaccharides, Golgi vesicles, and trans-Golgi network (TGN) of toxin-treated leaves of the susceptible cultivar, but not at Golgi vesicles and TGN of water-treated ones. The cis-, medial and trans-Golgi stacks of toxin-treated leaves of the susceptible cultivar were negative for the antibody. The results showed that the polysaccharides, Golgi vesicles and TGN contained abundant β-1,3-D-glucan and that the glucan was transported from the Golgi apparatus via Golgi vesicles to the modified sites in cells of toxin-treated leaves of the susceptible cultivar. Received 7 March 2002/ Accepted in revised form 10 June 2002  相似文献   

5.
ABSTRACT Host-specific toxins are produced by three pathotypes of Alternaria alternata: AM-toxin, which affects apple; AK-toxin, which affects Japanese pear; and AAL-toxin, which affects tomato. Each toxin has a role in pathogenesis. To facilitate molecular genetic analysis of toxin production, isolation of toxin-deficient mutants utilizing ectopic integration of plasmid DNA has been attempted. However, the transformation frequency was low, and integration events in most transformants were complicated. Addition of a restriction enzyme during transformation has been reported to increase transformation frequencies significantly and results in simple plasmid integration events. We have, therefore, optimized this technique, known as restriction enzyme-mediated integration (REMI), for A. alternata pathotypes. Plasmid pAN7-1, conferring resistance to hygromycin B, with no detectable homology to the fungal genome was used as the transforming DNA. Among the three restriction enzymes examined, HindIII was most effective, as it increased transformation frequency two-to 10-fold depending on the pathotype, facilitating generation of several hundred transformants with a 1-day protocol. BamHI and XbaI had no significant effect on transformation frequencies in A. alternata pathotypes. Furthermore, the transforming plasmid tended to integrate as a single copy at single sites in the genome, compared with trials without addition of enzyme. Libraries of plasmid-tagged transformants obtained with and without addition of restriction enzyme were constructed for the tomato pathotype of A. alternata and were screened for toxin production. Three AAL-toxin-deficient mutants were isolated from a library of transformants obtained with addition of enzyme. These mutants did not cause symptoms on susceptible tomato, indicating that the toxin is required for pathogenicity of the fungus. Characterization of the plasmid integration sites and rescue of flanking sequences are in progress.  相似文献   

6.
AK-toxin I caused plasma membrane modifications with plasma membrane-derived membrane fragments only in sensitive Japanese pear tissues. H2O2 generation was abundant in both the membrane fragments and the plasma membranes of the toxin-treated sensitive tissues. Whether lipid peroxidation was induced in plasma membranes of the toxin-treated sensitive tissues was examined biochemically and histochemically. Lipid peroxidation was caused only in the toxin-treated sensitive tissues or the toxin-treated plasma membrane-enriched fractions from sensitive young pear fruits. The results indicated that the peroxidation was probably induced by reactive oxygen species in the modified plasma membranes by action of toxin, suggesting that peroxidation is closely associated with plasma membrane modifications.  相似文献   

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8.
为了解橡胶树2种炭疽病菌的侵染结构发育分化过程,采用平板菌落生长速率法测定了3株胶孢炭疽菌Colletotrichum gloeosporioides和3株尖孢炭疽菌C.acutatum的菌丝生长速率,测量其分生孢子大小,显微观察2种炭疽菌在疏水表面诱导下侵染结构的发育分化过程。结果表明,胶孢炭疽菌菌丝生长速率为0.96~1.36 cm/d,显著高于尖孢炭疽菌的菌丝生长速率0.72~0.89 cm/d,但二者分生孢子大小无显著差异。在疏水表面诱导下,2种炭疽菌分生孢子在接种2~6 h后开始萌发,12 h孢子萌发率为71.70%~88.05%,13~16 h开始分化附着胞,24 h附着胞形成率为48.99%~70.74%,36 h菌丝诱发形成大量附着枝,48 h后分生孢子产生的次生菌丝也可诱发形成附着枝,附着枝呈圆形、姜瓣形、梨形或不规则形。分生孢子极易产生,可在菌丝顶端成簇或菌丝侧面排列产生,也可由分生孢子形成的芽管产生,或在芽管分化附着胞过程分枝形成分生孢子;附着胞多着生于芽管顶端,少数附着胞顶端可继续萌发类似短芽管结构,再次分化形成可黑色化的次级附着胞。表明橡胶树2种炭疽菌不同菌株间分生孢子萌发时间、孢子萌发率、附着胞形成时间和形成率有一定差异,但种间无明显差异;橡胶树炭疽菌分生孢子极易形成,在疏水表面容易分化形成附着胞和附着枝,说明具有极强的适生性。  相似文献   

9.
On the basis of mycelial compatibility, the population structure of Valsa ceratosperma, the causal fungus of Valsa canker on broadleaf trees, was investigated in apple and pear orchards in Japan. Field strains of V. ceratosperma from a single canker on trunks or scaffold limbs belonged to different mycelial compatibility groups. Thus, the population structure of this fungus was complex in most orchards. Because mycelia of strains originating from different conidia from the same pycnidium were compatible, infection by this fungus is thought to be ascospores.  相似文献   

10.
The inoculum sources of ascospores of Pleospora allii and of conidia of its anamorph Stemphylium vesicarium were investigated in relation to the brown spot disease epidemiology on pear. Dead and living leaves of three pear varieties (Abate Fétel, Conference and William), seven grasses (Poa pratensis, Festuca rubra, Festuca ovina, Lolium perenne, Digitaria sanguinalis and Setaria glauca) and Trifolium repens, which are used in pear orchard lawns, were inoculated with conidia of Stemphylium vesicarium virulent on pear and incubated under controlled-environment. Stemphylium vesicarium was always re-isolated from dead leaves of the considered plants, but not from symptomless green or yellowish living leaves. The fungus was occasionally re-isolated from leaf segments showing unspecific necrosis. Inoculation of pear leaves with isolates from grasses demonstrated that the fungus did not lose pathogenicity. Pseudothecia, ascospores and conidia were produced on all the dead inoculated leaves; differences between specimens were found for phenology of pseudothecia, their density and size, and for the number of conidia produced. Pseudothecia were produced faster in the lawn species than in pear leaves, and their density was higher, especially for S. glauca, L. perenne and P. pratensis. Ascospore maturation and ejection was more concentrated for the pseudothecia developed on pear leaves than for those on F. ovina and S. glauca. All the lawn species produced more conidia than pear leaves.  相似文献   

11.
Fungi inhabiting Japanese pear were isolated from internal tissues of cv. Nijisseiki, and culture filtrates (CFs) of 100 isolates were evaluated for their inhibitory activity against infection by Alternaria alternata Japanese pear pathotype. CFs of 11 isolates inhibited lesion formation on the pear by the pathogen. Among these isolates, CFs of five isolates inhibited spore germination. CFs of the six other isolates inhibited appressorial formation, infection hypha formation, AK-toxin production, or a combination of these actions. Analysis of sequence homology in the rDNA ITS1 regions of these isolates showed that most isolates had high homology with some fungal endophytes.  相似文献   

12.
In order to explore the molecular mechanisms of virulence and genetic variance of Curvularia lunata in maize, an ATMT (Agrobacterium tumefaciens-mediated transformation) system was established in order to create a wide range of insertional transformants of C. lunata. Our results showed that the germinating conidia were the ideal starting material for transformation. Based on our optimised transformation condition, the transformation efficiency of C. lunata with T-DNA was improved greatly, and the average transformation frequency was as high as 84 ± 5 transformants per 1 × 106 germlings. Southern blotting results of 39 randomly-selected transformants showed a unique hybridisation pattern and predominant single-copy insertions. An ATMT library containing approximate 3000 transformants was generated, and four types of transformants were screened in terms of growth rate, sporulation, mycelial pigmentation, and toxin production in vitro. This library will be used to identify genes involved in the virulence of the pathogen.  相似文献   

13.
The Japanese pear pathotype of Alternaria alternata, a toxin-dependent necrotrophic pathogen, causes black spot of Japanese pear by producing the host-specific AK-toxin. Pre-inoculation with nonpathogenic A. alternata or pretreatment with an elicitor prepared from A. alternata reduced disease symptoms caused by the pathogen. Salicylic acid- and jasmonic acid-dependent signaling pathways are not involved in the induced resistance to infection by the pathogen. The expression of multiple defense-related genes in Japanese pear leaves inoculated with nonpathogenic A. alternata was examined using suppression subtractive hybridization. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank database as accessions DC993229–DC993535.  相似文献   

14.
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15.
A study was conducted to determine the feasibility of using sclerotia ofSclerotinia sclerotiorum for producing conidia ofConiothyrium minitans in liquid culture. The medium (SST) was made of water containing 2.0, 1.5, 1.0 or 0.5% (w/v) ground sclerotia ofS. sclerotiorum and 100 μgl −1 thiamine hydrochloride (HCl). One ml of conidial suspension (2 × 107 conidia ml−1) ofC. minitans LRC 2534 was inoculated into 100 ml of SST medium or control (thiamine HCl in water) and incubated at 20 ± 2°C on a shaker at 200 rpm. Subsamples were removed periodically and examined under a compound microscope. Conidia in the SST media germinated within 24 h, developed into branched hyphae within 48 h, produced pycnidia after 3–4 days, and the pycnidia released mature conidia after 7 days. Production of conidia varied with the concentration of sclerotia in the SST medium. It was lower (3.6 × 106 conidia ml−1) at 0.5% but higher (1.2 × 108 conidia ml−1) at 2%. The new conidia were viable and the colonies developing from them showed the original morphological characteristics. It was concluded that using SST liquid medium as a substrate for mass production of conidia ofC. minitans has potential for use in commercial development of this mycoparasite as a biocontrol product. http:www.phytoparasitica.org posting Jan. 23, 2007.  相似文献   

16.
Experiments were conducted to determine the effects of temperature, relative humidity (RH) and duration of wetness period on in vitro germination of conidia and infection of detached pear leaves by Venturia nashicola , the causal agent of pear scab. Conidia germinated only in near-saturation humidity (RH > 97%). The final percentage germination (24 h after inoculation) at 100% RH without free water was less than half that in free water. Conidia germinated over the range of temperatures tested (5–30°C); the optimum temperature for germination was ≈21°C. Changes in percentage germination of conidia over time were fitted by logistic models at each individual temperature. Polynomial models satisfactorily described the relationships between two (rate and time to 50% of maximum germination) of the three logistic model parameters and temperature. The minimum length of the wetness period for successful infection of detached pear leaves by conidia was observed at several temperatures. The shortest length of wetness period required for infection was 7 h at 22°C. Two polynomial models fitted well the relationship between the minimum wetness duration required for infection, and temperature.  相似文献   

17.
Rice false smut disease caused by the ascomycete fungus Villosiclava virens (Clavicipitaceae) reduces rice yield worldwide. It invades rice panicles and forms dark‐green false smut balls composed of thick‐walled conidia. Although the infection process during the booting stage is well studied, its infection route before this is unclear. It was hypothesized that the thick‐walled conidia in soil penetrate rice roots, and the fungus latently colonizes roots and tiller buds at the vegetative stage. This hypothesis was tested using species‐specific detection methods. First, real‐time PCR with species‐specific primers and probe was used to estimate thick‐walled conidial number in the paddy field soil. Secondly, nested PCR with species‐specific primers showed that fungal DNA was detected in roots and shoot apices of rice plants in the vegetative stage. Thirdly, colourimetric in situ hybridization with a species‐specific oligonucleotide probe targeting 18S rRNA suggested that sparse mycelia or tightly condensed mycelia were present on the external surface of tiller buds enveloped by juvenile leaf sheaths at the vegetative stage. Thin hyphae were found around leaf axils at the surface of elongated stems at the heading stage, and the fungal hyphae grew in the rice root tissues. In addition, it was demonstrated that eGFP‐tagged transformants of the fungus invaded rice roots and colonized the surface of roots and leaf sheaths under artificial conditions.  相似文献   

18.
The activity of Trichoderma harzianum in the spermosphere and rhizosphere of different plant species was studied by use of a beta-glucuronidase (GUS) transformant (strain T3a). Hereby, direct observation of micro-habitats supporting metabolic activity of T. harzianum is reported. Germination of conidia and mycelial growth were not supported by exudates from healthy roots of various ages. Instead, growth and activity of T. harzianum depended on access to dead organic substrates such as seed coats, decaying roots, and wounds, including those caused by infecting pathogens. A correlation between the GUS activity of T. harzianum and the biomass of Pythium ultimum in infected roots was established. On the basis of our observations, we suggest that the biocontrol ability of T. harzianum involves competition with the pathogen for substrates including the seed coat, and wounded or infected root tissue.  相似文献   

19.
Light, scanning electron and fluorescent microscopy were used to observe the infection process of Botrytis elliptica on leaves of oriental lily (cv. Star Gazer). At 20 °C and 100% relative humidity, conidia germinated on both adaxial and abaxial foliar surfaces, but germ tubes failed to invade epidermal cells on the adaxial surface. On abaxial surfaces, short (< 20 m) swollen germ tube appressoria penetrated through stomatal openings (19%), through the epidermis near guard cells (52%), or directly through epidermal cells (29%). Esterase activity was detected on germ tubes and conidia after 6 h of incubation, and deformation of the cuticle on abaxial surfaces of lily was observed surrounding infection sites. By 3 h after inoculation, almost 70% of the conidia had germinated, but no penetration was observed. At 6 h after inoculation, almost one-third of germinated conidia had penetrated epidermal cells, and water-soaked lesions were associated with 20% of the penetrations. By 9 h after inoculation, approximately 60% of the germinated conidia had penetrated plant tissues, and water-soaked lesions were associated with 60% of the infections. Fluorescent microscopy with a specific fungal stain allowed assessment of successful infection and visualization of sub-epidermal hyphae. We conclude that penetration of abaxial foliar surfaces of oriental lilies by B. elliptica occurs via short swollen germ tube appressoria mostly near stomata.  相似文献   

20.
BACKGROUND: Testing the compatibility of chemical pesticides and fungal biocontrol agents is necessary if these two agents are to be applied together in the integrated management of plant pests and diseases. In this study, the fungicides azoxystrobin (a strobilurin) and flutriafol (a triazole) were tested in vitro for their effects on germination of conidia and mycelial growth of Beauveria bassiana (Bals.) Vuill. and in bioassay for their effect on fungal activity against Tetranychus urticae Koch. The fungicides were tested at three different concentrations [recommended rate for field use (1 × X) and the dilutions 10?1× X and 10?2× X]. RESULTS: Flutriafol inhibited growth of mycelia and germination of the fungal conidia at all concentrations tested in vitro, and also reduced the efficacy of B. bassiana in bioassays against mites. The inhibitive effect of azoxystrobin in vitro varied with the concentration applied. A significant effect was observed at 1 × X and 10?1× X concentrations on both the germination of conidia and mycelia growth. At 10?2× X concentration, azoxystrobin showed little effect on B. bassiana. However, when this fungicide was tested in bioassays, none of the concentrations reduced B. bassiana activity against mites. CONCLUSION: Azoxystrobin was most compatible with B. bassiana, while flutriafol was the most harmful. Further studies are required to confirm the negative effect of flutriafol on B. bassiana activity. Copyright © 2010 Society of Chemical Industry  相似文献   

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