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1.
Trichoderma (T. asperellum-203, 44 and GH11; T. atroviride-IMI 206040 and T. harzianum-248) parasitism on Meloidogyne javanica life stages was examined in vitro. Conidium attachment and parasitism differed beween the fungi. Egg masses, their derived eggs and second-stage juveniles (J2) were parasitized by Trichoderma asperellum-203, 44, and T. atroviride following conidium attachment. Trichoderma asperellum-GH11 attached to the nematodes but exhibited reduced penetration, whereas growth of T. harzianum-248 attached to egg masses was inhibited. Only a few conidia of the different fungi were attached to eggs and J2s without gelatinous matrix; the eggs were penetrated and parasitized by few hyphae, while J2s were rarely parasitized by the fungi. The gelatinous matrix specifically induced J2 immobilization by T. asperellum-203, 44 and T. atroviride metabolites that immobilized the J2s. A constitutive-GFP-expressing T. asperellum-203 construct was used to visualize fungal penetration of the nematodes. Scanning electron microscopy revealed the formation of coiling and appressorium-like structures upon attachment and parasitism by T. asperellum-203 and T. atroviride. Gelatinous matrix agglutinated T. asperellum-203 and T. atroviride conidia, a process that was Ca2+-dependent. Conidium agglutination was inhibited by carbohydrates, including fucose, as was conidium attachment to the nematodes. All but T. harzianum could grow on the gelatinous matrix, which enhanced conidium germination. A biomimetic system based on gelatinous-matrix-coated nylon fibers demonstrated the role of the matrix in parasitism: T. asperellum-203 and T. atroviride conidia attached specifically to the gelatinous-matrix-coated fibers and parasitic growth patterns, such as coiling, branching and appressoria-like structures, were induced in both fungi, similarly to those observed during nematode parasitism. All Trichoderma isolates exhibited nematode biocontrol activity in pot experiments with tomato plants. Parasitic interactions were demonstrated in planta: females and egg masses dissected from tomato roots grown in T. asperellum-203-treated soil were examined and found to be parasitized by the fungus. This study demonstrates biocontrol activities of Trichoderma isolates and their parasitic capabilities on M. javanica, elucidating the importance of the gelatinous matrix in the fungal parasitism.  相似文献   

2.
Sixteen isolates belonging to 11 species of Trichoderma (T. asperellum, T. ceramicum, T. andinensis, T. orientalis, T. atroviride, T. viridescens, T. brevicompactum, T. harzianum, T. virens, T. koningii and T. koningiopsis) were evaluated for biological control of potato (Solanum tuberosum) stem rot caused by Sclerotinia sclerotiorum. In dual culture tests, all antagonists significantly reduced sclerotia formation, and were able to inhibit radial growth of the pathogen. Growth inhibition by production of volatile and non-volatile inhibitors was also measured in in vitro tests. In screening the most efficient species of Trichoderma, establishment of mycelium on sclerotia and sclerotia lysis were also considered as important biocontrol qualities. Excluding T. asperellum, T. brevicompactum, T. andinensis and T. harzianum, all tested Trichoderma species were able to lyse sclerotia. The sclerotia-destroying species of Trichoderma and one isolate of Talaromyces flavus were tested in greenhouse tests and during 2 years of field experimentation during the 2007 and 2008 cropping seasons. After one aerial application of spore suspension in greenhouse trials, T. koningii, T. virens, T. ceramicum and T. viridescens were the most effective bio-agents and reduced significantly disease severity, and the least biocontrol efficacy was observed in T. flavus. Under field conditions and after five soil and foliar applications of spore suspension, all tested antagonists reduced significantly disease incidence. T. viridescens followed by T. ceramicum showed the best results. T. flavus and T. orientalis were less effective than other tested antagonists in both field trials.  相似文献   

3.
Trichoderma spp. are common soil fungi used as biocontrol agents due to their capacity to produce antibiotics, induce systemic resistance in plants and parasitize phytopathogenic fungi of major agricultural importance. The present study investigated whether colonization of Arabidopsis thaliana seedlings by Trichoderma atroviride affected plant growth and development. Here it is shown that T. atroviride promotes growth in Arabidopsis. Moreover, T. atroviride produced indole compounds in liquid cultures. These results suggest that indoleacetic acid-related indoles (IAA-related indoles) produced by T. atroviride may have a stimulatory effect on plant growth. In addition, whether colonization of Arabidopsis roots by T. atroviride can induce systemic protection against foliar pathogens was tested. Arabidopsis roots inoculation with T. atroviride provided systemic protection to the leaves inoculated with bacterial and fungal pathogens. To investigate the possible pathway involved in the systemic resistance induced by T. atroviride, the expression profile of salicylic acid, jasmonic acid/ethylene, oxidative burst and camalexin related genes was assessed in Arabidopsis. T. atroviride induced an overlapped expression of defence-related genes of SA and JA/ET pathways, and of the gene involved in the synthesis of the antimicrobial phytoalexin, camalexin, both locally and systemically. This is the first report where colonization of Arabidopsis roots by T. atroviride induces the expression of SA and JA/ET pathways simultaneously to confer resistance against hemibiotrophic and necrotrophic phytopathogens. The beneficial effects induced by the inoculation of Arabidopsis roots with T. atroviride and the induction of the plant defence system suggest a molecular dialogue between these organisms.  相似文献   

4.
A member of plant growth-promoting rhizobacteria, Bacillus megaterium YMF3.25, was demonstrated to be an efficient biocontrol agent (BCA) against root-knot nematode Meloidogyne incognita. Results from three-compartmented Petri dish tests and a pot experiment indicated that the bacterial culture could significantly inhibit the hatch of eggs and reduce infection of the nematode through production of nematicidal volatiles. After analysis by gas chromatograph/mass spectrometer and confirmation with commercial pure compounds, the nematicidal volatiles produced by the bacterium were characterised to include mainly the benzeneacetaldehyde, 2-nonanone, decanal, 2-undecanone and dimethyl disulphide, which were active against to both juveniles and eggs at the concentration of 0.5 mmol. Six compounds (phenyl ethanone, nonane, phenol, 3,5-dimethoxy-toluene, 2,3-dimethyl- butanedinitrile and 1-ethenyl-4-methoxy- benzene) with nematicidal activityies of 30%–63% also contributed to nematicidal efficacy of the bacterium.  相似文献   

5.
The antifungal activities of hyoscyamine and scopolamine, major alkaloids extracted from the desert plant Hyoscyamus muticus, against two rice pathogens, Magnaporthe oryzae and Rhizoctonia solani, were studied. The minimum inhibitory concentration of hyoscyamine that resulted in distinctive inhibition (MIC50) was 1 μg/ml for both fungi. Exposure to hyoscyamine caused the leakage of electrolytes from the mycelia of both fungi. Hyoscyamine (>1 μg/ml) irreversibly delayed or inhibited conidial germination and appressorium formation in M. oryzae grown on polystyrene plates. Hyoscyamine effectively inhibited the attachment of conidia to the surface of rice (Oryza sativa) leaves and inhibited appressorium formation on the leaves. A high concentration of scopolamine (1000 μg/ml) also delayed or inhibited conidial germination in M. oryzae, but conidial germination was restored after washing the conidia with water. Antifungal activity of hyoscyamine was reduced by scopolamine. Magnaporthe oryzae infection was significantly suppressed (by >95%) in leaves of intact rice plants treated with hyoscyamine (10 μg/ml). Moreover, 10 μg hyoscyamine/ml significantly reduced the disease severity index for sheath blight to ≤0.2, when compared with the disease index of control plants (>7.0). Hyoscyamine (>20 μg/ml) completely inhibited sclerotial germination and development of R. solani by delaying the initiation, maturation, and melanization of the sclerotia. These results suggest that tropane alkaloids may be useful for controlling blast and sheath blight diseases of rice and for studying the mechanisms that regulate conidial germination in M. oryzae and sclerotial germination and development in R. solani.  相似文献   

6.
Inheritance of the resistance to nematodes has been studied on many different crops, however to our knowledge, no data are available for guava species. The basic genetic resistance parameters to Meloidogyne enterolobii are estimated in the current research in order to guide the development of genotypes resistant to the pathogen. The parental plants, F1 and F2 from a Psidium guajava x P. guineense cross were assessed for the presence or absence of galls and for the number of eggs and juveniles in the root system at the 120th and 240th days after inoculation with 10,000 eggs and juveniles of the nematode. At the age of nine years, the P. guineense plant remained without nematode attack symptoms, whereas the maternal plant was destroyed by the pathogen. The F1 generation showed 270 plants with reproduction factor (RF) <0.322, and there were tiny galls in only 16 plants. The segregation for the presence or absence of galls in the root system in generation F2 was 9:7, wherein the χ2 values were 0.78 and 2.66, respectively, at the 120th and 240th days after inoculation, whereas the segregation for RF was 15:1, wherein the χ2 values were 2.76 and 1.18, respectively, at the 120th and 240th days. These results indicate epistatic interaction between two genes: in RF < 1 only one allele sets the resistance to the pathogen. The broad sense heritability of RF, estimated to the two assessments was 0.97, and it also indicates a simple inheritance of resistance to M. enterolobii.  相似文献   

7.
The interaction between Alternaria dauci and two carrot cultivars differing in their resistance to leaf blight was investigated by microscopy. The fungal development between 1 and 15 days post-inoculation was quite similar in the susceptible cv. Presto and the partially resistant cv. Texto: After conidial germination, leaf adhesion of the pathogen was achieved with mucilaginous filaments; hyphae penetrated the leaves directly with/without the formation of appressoria-like structures or via stomata; the fungus spread by epiphytic hyphae with hyphopodia and subcuticular mycelia. Intense necrotic plant cell reactions occurred under the fungal structures. At 21 days post-inoculation, typical features of fungal development were noted for each cultivar: growing hyphae emerged from stomata in cv. Presto, whereas conidiophores without conidia were observed in cv. Texto. Leaf tissues of both cultivars were strongly damaged and vesicle-like structures (assumed to be plant phenolics) were abundantly present between mesophyll cells. A real-time PCR method was developed for in planta quantification of A. dauci. Between 1 and 15 days post-inoculation, the fungal biomass was equivalent in the two cultivars and was about fourfold higher in cv. Presto than cv. Texto at 21 and 25 days post-inoculation. Taken together, our results indicated that A. dauci was able to colonize both cultivars in a similar manner during the first steps of the interaction, then fungal development in the partially resistant cultivar was restricted due to putative plant defence reactions. The results of this study enhance the overall understanding of infection processes in the A. dauci-carrot pathosystem.  相似文献   

8.
The rhizobacterium Paenibacillus macerans was grown in tryptic soy broth and after separating the cells by centrifugation the activity of fractions of the supernatant was tested against Meloidogyne exigua juveniles. From HPLC analyses and spectral data, the most active fractions were found to contain alanine, glutamic acid, glycine, histidine, threonine and valine, which were probably produced by bacterial hydrolysis of proteic nutrients. Amino acids from commercial sources were then assayed to confirm these results and to evaluate their potential for the control of nematodes. LC50 of 26 and 283 μg ml−1 were shown for the nematicide aldicarb and L-cysteine respectively when tested on M. exigua juveniles. At a concentration 38.4 times>LC50, the amino acid diminished the nematode population on coffee plants to values statistically equal to those obtained with aldicarb at a concentration 19.2 times>LC50.  相似文献   

9.
Root-knot nematodes (RKNs) are one of the most important biotic factors limiting crop productivity in many crop plants. The major RKN control strategies include development of resistant cultivars, application of nematicides and crop rotation, but each has its own limitations. In recent years, RNA interference (RNAi) has become a powerful approach for developing nematode resistance. The two housekeeping genes, splicing factor and integrase, of Meloidogyne incognita were targeted for engineering nematode resistance using a host-delivered RNAi (HD-RNAi) approach. Splicing factor and integrase genes are essential for nematode development as they are involved in RNA metabolism. Stable homozygous transgenic Arabidopsis lines expressing dsRNA for both genes were generated. In RNAi lines of splicing factor gene, the number of galls, females and egg masses was reduced by 71.4, 74.5 and 86.6%, respectively, as compared with the empty vector controls. Similarly, in RNAi lines of the integrase gene, the number of galls, females and egg masses was reduced up to 59.5, 66.8 and 63.4%, respectively, compared with the empty vector controls. Expression analysis revealed a reduction in mRNA abundance of both targeted genes in female nematodes feeding on transgenic plants expressing dsRNA constructs. The silencing of housekeeping genes in the nematodes through HD-RNAi significantly reduced root-knot nematode infectivity and suggests that they will be useful in developing RKN resistance in crop plants.  相似文献   

10.
Leaves of tomato and barley were inoculated with conidia of Blumeria graminis f. sp. hordei race 1 (R1) or Oidium neolycopersici (KTP-01) to observe cytological responses in search of resistance to powdery mildew. Both conidia formed appressoria at similar rates on tomato or barley leaves, indicating that no resistance was expressed during the prepenetration stage of these fungi. On R1-inoculated tomato leaves, appressoria penetrated the papillae, but subsequent haustorium formation was inhibited by hypersensitive necrosis in the invaded epidermal cells. On the other hand, KTP-01 (pathogenic to tomato leaves) successfully developed functional haustoria in epidermal cells to elongate secondary hyphae, although the hyphal elongation from some conidia was later suppressed by delayed hypersensitive necrosis in some haustorium-harboring epidermal cells. Thus, the present study indicated that the resistance of tomato to powdery mildew fungi was associated with a hypersensitive response in invaded epidermal cells but not the prevention of fungal penetration through host papilla.  相似文献   

11.
The feeder roots of pepper plants (cv. California Wonder) in Campo de Cartagena (southeast Spain) were found to be severely infected by Meloidogyne incognita. Morphometric traits, differential host test and DNA analysis based on PCR were used to characterize the nematode. Naturally and artificially infected pepper plants showed severe yellowing and stunting, with heavily deformed and damaged root systems. Root galls were spherical and commonly contained more than one female and egg masses with eggs. Typical giant cells with a granular cytoplasm and many hypertrophied nuclei were observed in histological preparations. The relationship between initial nematode population density (Pi) and pepper plant growth was tested in greenhouse experiments with inoculum levels that varied from 0 to 64 eggs and second-stage juveniles (J2) ml−1 soil. A Seinhorst model was fitted to plant height and top fresh weight data of inoculated and non-inoculated plants. The tolerance limit with respect to plant height and fresh top weight of pepper to M. incognita was estimated as 0.85 eggs and J2 ml−1 soil. The minimum relative values (m) for plant height and top fresh weight were 0.15 and 0.16, respectively, at Pi ≥ 64 eggs and J2 ml−1 soil. The maximum nematode reproduction rate (Pf/Pi) was 315.4 at an initial population density (Pi) of 4 eggs and J2 ml−1 soil. The obtained results could be used as a base to establish field experiments that allow strategies to prevent surpassing the threshold of nematodes in fields that are infested.  相似文献   

12.
The tomato pathotype of Alternaria alternata (A. arborescens) produces the dark brown to black pigment melanin, which accumulates in the cell walls of hyphae and conidia. Melanin has been implicated as a pathogenicity factor in some phytopathogenic fungi. Here, two genes of the tomato pathotype for melanin biosynthesis, ALM1 and BRM2-1, which encode a polyketide synthetase and a 1,3,8-trihydroxynaphthalene (THN) reductase, respectively, have been cloned and disrupted in the pathogen. The gene-disrupted mutants, alm1 and brm2-1, had albino and brown phenotypes, respectively. The wild-type and the mutants caused the same necrotic lesions on the leaves after inoculation with spores. These results suggest that melanin is unlikely to play a direct role in pathogenicity in the tomato pathotype A. alternata. Scanning electron microscopy revealed that the conidia of both mutants have much smoother surfaces in comparison to the wild-type. The conidia of those mutants were more sensitive to UV light than those of the wild-type, demonstrating that melanin confers UV tolerance.  相似文献   

13.
Trichoderma spp. are used as antagonists against different pathogens. Despite many possibilities of using Trichoderma as an antagonist, there are gaps in the knowledge of the interaction between Trichoderma, cassava and Scytalidium lignicola. This fungus causes cassava black root rot and is an inhabitant of the soil, so it is difficult to control. Antagonists may contribute to the possible induction of resistance of plants because, when exposed to such pathosystems, plants respond by producing antioxidative enzymes. The test for potential inhibition of growth of S. lignicola CMM 1098 in vitro was performed in potato-dextrose-agar with two Trichoderma strains T. harzianum URM3086 and T. aureoviride URM 5158. We evaluated the effect of the two selected Trichoderma to reduce the severity of cassava black root rot and shoots. Subsequently, the production of enzymes (ascorbate peroxidase, catalase, peroxidase and polyphenol oxidase) was evaluated in cassava plants. All two Trichoderma strains show an inhibition of the growth of S. lignicola CMM 1098. The most efficient was T. harzianum URM 3086, with 80.78% of mycelial growth inhibition. T. aureoviride URM 5158 was considered the best chitinase producer. All treatments were effective in reducing severity, especially treatments using Trichoderma. Cassava plants treated with T. aureoviride URM 5158 had the highest enzyme activity, especially peroxidase and ascorbate peroxidase. Trichoderma harzianum URM3086 and Trichoderma aureoviride URM 5158 were effective in reducing the severity of cassava black root rot caused by S. lignicola CMM 1098.  相似文献   

14.
Pyricularia oryzae (rice blast) conidial development at pre-penetration stage determines success or otherwise of infection inside the rice host plants. Studies on conidial germination and growth on the leaf surface in commercial rice (Oryza sativa) report differently, dependent upon host type and level of blast resistance. Although wild rice (O. australiensis) is known to be an alternative host of blast, the interaction between P. oryzae conidia and wild O. australiensis on its leaf surface has not been previously studied. We found significant (P?<?0.001) differences in conidial development between two blast isolates with different virulence in terms of conidial germination, germ tube growth and appressoria formation on both wild and cultivated rice. Conidial germination at 6 h post-inoculation (hpi) for the virulent isolate was significantly (P?<?0.001) delayed. Germ tubes of the avirulent isolate conidia grew significantly (P?<?0.001) faster and with significantly (P?<?0.001) longer germ tubes than from virulent conidia. Appressoria development for the virulent isolate was significantly (P?<?0.001) faster at its later growth stages of 12 and 18 hpi when approximately 100% of germ tubes formed appressoria. In contrast, formation rate of appressoria for the avirulent isolate was significantly (P?<?0.001) slower and only reached 76% of germ tubes forming appressoria. Appressoria formation on O. australiensis was significantly (P?<?0.001) greater than the formation on O. sativa for both virulent and avirulent P. oryzae at 12 hpi, a clear indication that host type influences the extent of appressoria formation.  相似文献   

15.
Among the factors affecting the quality and yield of garlic production, blue mold caused by -- Penicillium spp. -- is responsible for economical losses in many countries. Allicin, present in garlic bulbs, has been suggested as having antifungal activity against some Penicillium species. This study was conducted to evaluate the response of garlic accessions against Penicillium hirsutum infection and to compare this response with bulb allicin content. Twelve garlic accessions were inoculated with P. hirsutum, and assayed in greenhouse and growth chamber experiments. Plant growth parameters and the fungal production of conidia were evaluated. Significant differences were found among the accessions. Accessions Castaño and Morado were most resistant whereas AR-I-125 and Fuego were always severely affected by the disease. A low correlation was found (r = 0.17) between allicin content and tolerance, indicating that allicin is not the main factor involved in the resistance against P. hirsutum.  相似文献   

16.
The pathogenicity and reproductive fitness of Pratylenchus coffeae and Radopholus arabocoffeae from Vietnam on coffee (Coffea arabica) seedlings cv. Catimor were evaluated in greenhouse experiments. The effect of initial population densities (Pi = 0, 1, 2, 4, 8, 16, 32, 64, 128, and 256 nematodes per cm3 soil) was studied for both species at different days after inoculation (dai). The data were adjusted to the Seinhorst damage model Y = m + (1-m).zPi-T. Tolerance limit (T) for P. coffeae was zero for the height and the diameter of the coffee plants. For the diameter, the T-value for R. arabocoffeae was 25.6 for 30 and 60 dai and 12.8 for 90 and 120 dai. After 4 months T was zero. The low tolerance limits indicate that Arabica coffee is highly intolerant to both nematode species. At the end of the experiment (180 dai), all plants were infected and most were dead when inoculated with R. arabocoffeae at initial densities of 32, 64, 128 and 256 nematodes/cm3 soil. For P. coffeae plant death was already observed at the lowest inoculation densities. Growth of coffee was reduced at all inoculation levels for both species. Pratylenchus coffeae and R. arabocoffeae caused intense darkening of the roots, leaf chlorosis and a strong reduction of root and shoot growth. It was observed that P. coffeae mainly destroyed lateral roots rather than tap roots, whereas R. arabocoffeae reduced tap root length rather than the lateral roots. At the lowest inoculum densities, the reproduction factor of P. coffeae was 2.38 and 2.01 for R. arabocoffeae, indicating that arabica coffee is a host for both species. Plant growth as expressed by shoot height and shoot and root weight measured 60 dai was negatively correlated with nematode (both species) density as expressed by the geometric mean of nematode numbers at 30 and 60 dai.  相似文献   

17.
Experiments were conducted for 3 seasons, 2007–2008, 2008–2009 and 2009–2010 in a wheat field planted with a cultivar susceptible to powdery mildew in Langfang City, Hebei Province, China. Plants were inoculated with Blumeria graminis f. sp. tritici (Bgt) and conidia of Bgt in the air were trapped using volumetric spore samplers. Disease severity was recorded weekly. The relationships between airborne conidial concentrations and meteorological factors, as well as disease index were analyzed. Conidia were first detected about 20 days after inoculation in all three seasons, and then increased gradually with time. The highest conidial concentrations in the air were observed in mid-May 2008 and 2009 and late May 2010 at growth stage (GS) 10.5.4. The concentrations of Bgt conidia after inoculation (GS 5) to milky ripe (GS 11.1) in the air were positively correlated with temperature, solar radiation, and negatively with relative humidity and vapor pressure deficit (VPD). Prediction models of Bgt conidial concentrations in the air based on meteorological factors were constructed using multiple regression analysis. Time series analysis, using autoregressive integrated moving average (ARIMA) (p, d, q) models, showed that each of the three season’s data can be fitted with simple ARIMA (1, 0, 0) models. Conidial concentrations within the canopy were significantly higher than those above the canopy (P < 0.01). The weekly-accumulated mean hourly conidia per cubic metre of air significantly (P < 0.01) correlated with disease index in all three seasons.  相似文献   

18.
From the genome of a Japanese field isolate of the rice blast fungus, Magnaporthe oryzae, we newly identified Inago1 and Inago2 LTR retrotransposons. Both elements were found to be Ty3/gypsy-like elements whose copies were dispersed within the genome of Magnaporthe spp. isolates infecting rice and other monocot plants. Southern hybridization patterns of nine re-isolates derived from conidia of the strain Ina168 produced after a methyl viologen treatment were not changed, indicating that the insertion pattern of Inago elements is relatively stable.  相似文献   

19.
The greater wax moth Galleria mellonella L. (Lepidoptera: Pyralidae) is occasionally found in beehives and is a major pest of stored wax. Entomopathogenic fungi have recently received attention as possible biocontrol elements for certain insect pests. In this study, 90 isolates of Beauveria bassiana and 15 isolates of Metarhizium anisopliae were screened for proteases and lipases production. The results showed significant variations in the enzymatic action between the isolates. In the bioassay, the selected isolates evinced high virulence against the 4th instar of the G. mellonella larvae. The isolates BbaAUMC3076, BbaAUMC3263 and ManAUMC3085 realized 100% mortality at concentrations of 5.5 × 106 conidia ml−1, 5.86 × 105 conidia ml−1, and 4.8 × 106 conidia ml−1, respectively. Strong enzymatic activities in vitro did not necessarily indicate high virulence against the tested insect pest. The cuticle of the infected larvae became dark and black-spotted, indicating direct attack of fungus on the defense system of the insects. The LC50 values were 1.43 × 103, 1.04 × 105 and 5.06 × 104 for Bba3263AUMC, Bba3076AUMC and Man3085AUMC, respectively, and their slopes were determined by computerized probit analysis program as 0.738 ± 0.008, 0.635 ± 0.007 and 1.120 ± 0.024, respectively.  相似文献   

20.
Random insertional mutagenesis using a marker DNA fragment is an effective method for identifying fungal genes relevant to morphogenesis, metabolism, and so on. Agrobacterium tumefaciens-mediated transformation (AtMT) has long been used as a tool for the genetic modification of a wide range of plant species. Recent study has indicated that A. tumefaciens could transfer T-DNA not only to plant cells but also to fungal cells. In this study, AtMT was applied to Colletotrichum lagenarium for random insertional mutagenesis. We constructed a binary vector pBIG2RHPH2 carrying a hygromycin-resistant gene cassette between the right and left borders of T-DNA. Optimal co-cultivation of C. lagenarium wild-type 104-T with pBIG2RHPH2-introduced A. tumefaciens C58C1 led to the production of 150–300 hygromycin-resistant transformants per 106 conidia. Southern blot analysis revealed that T-DNA was mainly integrated at a single site in the genome and at different sites in transformants. The T-DNA inserts showed small truncations of either end, but the hygromycin-resistant gene cassette inside the T-DNA was generally intact. The mode of T-DNA insertion described above resulted in highly efficient gene recovery from the transformants by thermal asymmetrical interlaced-polymerase chain reaction. The fungal genomic DNA segments flanking T-DNA were identified from five of eight mutants that had defective melanin biosynthesis. The sequence from one of the segments was identical to that of the melanin biosynthesis gene PKS1 of C. lagenarium, which we previously characterized. These results strongly support our notion that AtMT is a possible tool for tagging genes relevant to pathogenicity in the plant pathogenic fungus C. lagenarium.  相似文献   

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