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1.
A real-time PCR assay was designed to quantify seed-borne infection of Pyrenophora graminea in barley (Hordeum vulgare). Conventional tests such as the freezing blotter method cannot distinguish P. graminea from the closely related P. teres. The seed infection threshold for P. graminea is lower than the one for P. teres and is therefore applied for both species although P. graminea may be absent. This results in unnecessary rejections of seed lots. PCR primers and a TaqMan probe were designed to target a P. graminea-specific DNA sequence. The potential of the real-time PCR assay for quantifying seed-borne infection of P. graminea was investigated by examining seed lots harvested from P. graminea-infected fields. The major part (84%) of the variation in the amount of P. graminea DNA measured by real-time PCR could be attributed to variation between seed lots while only about 8% was due to variation within seed lots. DNA quantities of P. graminea were positively correlated with seed infection incidence detected by the freezing blotter method as well as with the infection incidence of plants examined in the greenhouse. Both correlations were highly significant (P < 0.001) but the DNA quantities accounted only for 59% (R 2 = 0.59) and 56% (R 2 = 0.56), respectively, of the variation in the results obtained by the two conventional methods. Seed lots of varieties resistant to P. graminea contained considerable amounts of P. graminea DNA but showed no or only few leaf symptoms in the greenhouse test suggesting that the recommended seed infection thresholds could be raised for resistant varieties.  相似文献   

2.
Spot blotch, caused by Cochliobolus sativus (Ito & Kuribayashi) Drechs. ex Dastur, is one of the important diseases of wheat worldwide. The main objective of this study was to investigate the phenotypic and genotypic variability among C. sativus isolates from the hills and plains in Nepal. A total of 48 monoconidial isolates of C. sativus from the hills (n = 24 isolates) and plains (n = 24 isolates) in Nepal were analyzed for morphology, aggressiveness and genetic structure. C. sativus isolates were grouped into three categories on the basis of their colony texture and mycelia colour. Thirteen isolates from the hills and plains belonging to three morphological groups were randomly selected and evaluated for aggressiveness on eight wheat cultivars (Chirya 1, Chirya 7, Milan/Shanghai 7, SW 89–5422, PBW 343, BL 1473, BL 3036, and RR 21) at the seedling stage. Nonparametric analysis revealed that the isolates from the plains (median disease rating of 5) were significantly (P = 0.0001) more aggressive than the isolates from the hills (median disease rating of 3). A significant (P = 0.0001) isolate by cultivar interaction was demonstrated and the isolates from the same geographic region and morphological group displayed different degrees of aggressiveness on wheat cultivars tested. Combined IS-PCR and rep-PCR analyses revealed moderate gene diversity (H = 0.24 and 0.25 for the hills and plains, respectively). Low linkage disequilibrium (LD) value and non-significant (P = 0.001) population differentiation (G″ST = 0.05) were detected, indicating that isolates of C. sativus from the hills and plains in Nepal were genetically similar. Analysis of molecular variation (AMOVA) revealed low (7%) levels of genetic variation between the hill and plain populations, whereas >93% of genetic variation was found within populations. Overall, C. sativus isolates from Nepal are pathologically and genetically diverse, and such information will be useful in developing wheat cultivars resistant to C. sativus.  相似文献   

3.
Fusarium equiseti is prevalent in ginseng soil, straw mulch and in ginseng root tissues and is the cause of a root surface discolouration on ginseng grown in British Columbia. Population levels of the fungus in ginseng fields ranged from 3.8 × 103 cfu g−1 soil to 1.4 × 104 cfu g−1 soil and were highest at 0–5 cm soil depths compared to 10–15 cm. Soil population levels were negatively correlated with S content in soil and positively correlated with Zn levels. Barley or wheat straw added to soil significantly increased population levels under laboratory conditions. Mycelial growth in culture was highest at 26–30°C and at pH 7.2–7.8. Samples of flowers and berries, and harvested seed, contained DNA of F. equiseti detected using a Fusarium-specific DNA array and the fungus was isolated from these tissues on agar medium. A high degree of genetic variation in the EF-1 alpha gene sequence was present among 52 isolates of F. equiseti which originated from ginseng fields. At least seven clades were identified. Inoculum dispersal from straw mulch used in ginseng gardens can result in seed contamination by the fungus. In addition, fungal growth near the soil surface under warm summer conditions can result in infection and crown discolouration of ginseng roots.  相似文献   

4.
Beet necrotic yellow vein virus (BNYVV) is transmitted by Polymyxa betae to sugar beet, causing rhizomania disease. Resistance-breaking strains of BNYVV, overcoming single (Rz1) or double (e.g. Rz1  +  Rz2) major resistance genes in sugar beet have been observed in France and recently in the USA and Spain. To demonstrate if resistance-breaking is dependent on inoculum density, the inoculum concentration of BNYVV and P. betae in soil samples where resistance-breaking had been observed was estimated using the most probable number (MPN) method. The MPN-values obtained displayed highly significant differences with respect to the virus concentration in various soils and did not correlate with the ability to overcome resistance. Virus quantification in susceptible plants demonstrated that soils containing resistance-breaking isolates of BNYVV did not produce higher virus concentrations. The MPN assay was repeated with Rz1  +  Rz2 partially-resistant sugar beets to see if the resistance-breaking is concentration-dependent. There was no correlation between soil dilution and increased virus concentration in Rz1  +  Rz2 plants produced by BNYVV resistance-breaking strains. Determination of the absolute P. betae concentration by ELISA demonstrated that all resistance-breaking soil samples contained elevated concentrations. However, the calculation of the proportion of viruliferous P. betae did not show a positive correlation with the resistance-breaking ability. Finally resistance-breaking was studied with susceptible, Rz1 and Rz1  + Rz2 genotypes and standardised rhizomania inoculum added to sterilised soil. Results from these experiments supported the conclusion that resistance-breaking did not correlate with virus concentration or level of viruliferous P. betae in the soil.  相似文献   

5.
A laboratory assay was designed to determine the insecticidal efficacy of Beauveria bassiana (Balsamo) Vuillemin (Hyphomycetes: Moniliales) and diatomaceous earth (Diafil 610) against Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae). The fungus B. bassiana was applied at 2.23 × 107, 2.23 × 108 and 2.23 × 109 conidia kg−1 of wheat individually as well as mixed with 200 and 400 ppm of Diafil 610. The conditions for the trials were 30 ± 2oC with 55% r.h. and the counts for mortality were made after 8, 16 and 24 d. All the dead adults were removed after each count and the vials were kept for the next 60 d to assess the emergence of the F1 generation. The findings from these studies proved that the extended exposure interval and the highest combined dose rate of the entomopathogenic fungus and the diatomaceous earth gave the maximum mortality of the beetles. The emergence of the progeny was also highly suppressed where the maximum dose rate of the synergized treatments was applied. The rate of mycosis and sporulation in the cadavers of R. dominica was maximum where the low dose rates of B. bassiana were applied.  相似文献   

6.
The yeast Pichia anomala strain K was selected in Belgium from the apple surface for its antagonistic activity against post-harvest diseases of apples. The efficacy of this strain against P. expansum was evaluated in the laboratory in three scenarios designed to mimic practical conditions, with different periods of incubation between biological treatment, wounding of fruit surface, and pathogen inoculation. Higher protection levels and higher final yeast densities were obtained when the applied initial concentration was 1 × 108 cfu ml−1 than when it was only 1 × 105 cfu ml−1. The protection level correlated positively with the yeast density determined in wounds and was influenced by apple surface wetness. In orchard trials spanning two successive years, biological treatment against P. expansum, based on a powder of P. anomala strain K (1 × 107 cfu ml−1), β-1,3-glucans (YGT 2 g l−1), and CaCl2.2H20 (20 g l−1), was applied to apples pre- or post-harvest under practical conditions and its effect compared with standard chemical treatments. The first year, the highest reduction (95.2%) against blue decay was obtained by means of four successive fungicide treatments and the next-highest level (87.6%) with pre-harvest high-volume spraying of the three-component mixture 12 days before harvest. The second year, the best results were obtained with post-harvest Sumico (carbendazim 25% and diethofencarb 25%) treatment and post-harvest biological treatment, both by dipping the apples, 88.3 and 56.3% respectively. A density threshold of 1 × 104 cfu cm−2 of strain K on the apple surface seemed to be required just after harvest for high protective activity, whatever the method and time of application. In the case of pre-harvest biological treatments, variations in meteorological conditions between the 2 years may have considerably affected strain K population density and its efficacies.  相似文献   

7.
Resistance to the fungicide boscalid in laboratory mutants of Botryotinia fuckeliana (Botrytis cinerea) was investigated. The baseline sensitivity to boscalid was evaluated in terms of colony growth (EC50 = 0.3–3 μg ml−1; MIC = 10–30 μg ml−1) and conidial germination (EC50 = 0.03–0.1 μg ml−1; MIC = 1–3 μg ml−1) tests. Mutants were selected in vitro from wild-type strains of the fungus on a fungicide-amended medium containing acetate as a carbon source. Mutants showed two different levels of resistance to boscalid, distinguishable through the conidial germination tests: low (EC50 ∼ 0.3 μg ml−1, ranging from 0.03 to 1 μg ml−1; MIC > 100 μg ml−1) and high (EC50 > 100 μg ml−1) resistance. Analysis of meiotic progeny from crosses between resistant mutants and sensitive reference strains showed that resistant phenotypes were due to mutations in single major gene(s) inherited in a Mendelian fashion, and linked with both the Daf1 and Mbc1 genes, responsible for resistance to dicarboximide and benzimidazole fungicides, respectively. Gene sequence analysis of the four sub-units of the boscalid-target protein, the succinate dehydrogenase enzyme, revealed that single or double point mutations in the highly conserved regions of the iron-sulphur protein (Ip) gene were associated with resistance. Mutations resulted in proline to leucine or phenylalanine replacements at position 225 (P225L or P225F) in high resistant mutants, and in a histidine to tyrosine replacement at position 272 (H272Y) in low resistant mutants. Sequences of the flavoprotein and the two transmembrane sub-units of succinate dehydrogenase were never affected.  相似文献   

8.
Wild type (WT) field isolates of Bremia lactucae failed to germinate in vitro or infect lettuce leaves in the presence of CAA (carboxylic acid amide) fungicides. Minimal inhibitory concentrations (MIC) for mandipropamid, dimethomorph, benthiavalicarb and iprovalicarb were 0.005, 0.5, 0.5 and 5 μg ml−1, respectively. Mutagenesis experiments showed that spores exposed to EMS (ethyl methane sulphonate) or UV irradiation (254 nm) could infect lettuce leaves in the presence of up to 100 μg ml−1 CAA. The proportion of infected leaves relative to the number of spores inoculated (infection frequency) was inversely related to the concentration of CAA used, ranging between 0 and 160 per 1 × 106 spores. Resistant mutants (RM) lost their resistance within 1–14 reproduction cycles on CAA-treated plants. Crosses were made between RMxWT isolates and RMxRM isolates with an attempt to obtain stable homozygous resistant off-springs. Such crosses yielded few resistant but unstable progeny isolates. Mutagenic treatments given to hybrid isolates also failed to produce stable resistance. Previous gene sequencing data showed that stable resistance to CAAs is based on a single SNP in the cellulose synthase 3 (CesA3) gene of Plasmopara viticola. Therefore, we sequenced a 582 bp DNA fragment of Ces3A of WT, RM and hybrid isolates of B.lactucae. No mutation in this gene fragment was found. We conclude that mutagenic agents like EMS or UV may induce resistance to CAA in Bremia lactucae but this resistance is not stable and not linked to mutations in CesA3 gene.  相似文献   

9.
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   

10.
A nonpathogenic strain of Agrobacterium vitis VAR03-1 was tested as a biological control agent against crown gall of grapevine (Vitis vinifera L.). A mixture of the nonpathogenic strain VAR03-1 and a tumorigenic strain G-Ag-27 of A. vitis at cell ratios of 1 : 1, 3 : 1, 9 : 1, and 99 : 1 significantly inhibited gall formation and size on stems of tomato (Lycopersicon esculentum Mill.). Strain VAR03-1 also inhibited gall formation on stems of both tomato and grapevine at a 1 : 1 cell ratio with several tumorigenic A. vitis strains isolated from different fields of grapevine in Japan. In biological control tests, when roots of grapevine and tomato seedlings were soaked in a cell suspension of strain VAR03-1 for 24 h before a 1-h soaking in a cell suspension of the pathogen and subsequent planting in pots of infested soil, strain VAR03-1 significantly reduced the incidence of gall formation on both plants.  相似文献   

11.
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12.
Apple proliferation (AP), caused by ‘Candidatus Phytoplasma mali’, is an economically important disease affecting many apple-growing areas in Europe. A new TaqMan real-time PCR assay was established for absolute quantification of ‘Ca. P. mali’ by using a single-copy gene of the host plant as a reference, which is amplified with the pathogen DNA in a single-tube reaction. Normalised estimates of phytoplasma concentration are ultimately expressed as the number of phytoplasma cells per host plant cell. The assay was used to monitor the ‘Ca. P. mali’ titre over the course of two growing seasons in roots and branches of symptomatic and asymptomatic but AP-positive apple trees. All 252 root samples from symptomatic and asymptomatic trees tested positive, with an average number of 59.8 ± 5.68 (standard error) and 55.1 ± 9.83 ‘Ca. P. mali’ per host cell, respectively. From the 378 shoot samples analysed, 81% of the symptomatic and only 20% of the asymptomatic samples were AP-positive with an average number of 9.4 ± 1.04 and 0.7 ± 0.13 ‘Ca. P. mali’ per host cell, respectively. This strengthens evidence that not the pathogen occurrence alone but the presence of a certain quantity of ‘Ca. P. mali’ in the aerial tree sections is involved in symptom expression. In addition, pronounced seasonality of the phytoplasma concentration was found, not only in branches, but also for the first time in roots of symptomatic and asymptomatic apple trees. Highest phytoplasma levels in roots were detected from December to May.  相似文献   

13.
The vertical distribution of leaf blast lesions caused by the fungus Pyricularia grisea was studied to estimate the degree of leaf blast suppression in rice multilines in experimental paddy fields for 4 years. Leaf blast in 1 : 1 and 1 : 3 mixtures of susceptible rice cultivar Sasanishiki and its resistant near-isogenic line, Sasanishiki BL7, developed slower than that in pure stands of Sasanishiki. The average distance of lesions on leaves from the ground in the 1 : 3 mixtures was significantly lower than that in the pure stands at the end of leaf blast epidemics (at booting stage). This result shows that the distribution of leaf blast lesions in the upper layer differs between the susceptible pure stands and the 1 : 3 mixtures at the end of leaf blast epidemics.  相似文献   

14.
Fusarium graminearum is a common agent causing Fusarium head blight (FHB) on wheat throughout the world. Aggressiveness is crucial for understanding the interaction between host-pathogen in the FHB-wheat system. In this paper, we modified and validated the Petri-dish test originally described by Mesterhazy (Phytopathologische Zeitschrift 93:12–25, 1978) to quantify the aggressiveness of 25 F. graminearum strains using four durum wheat cultivars with different resistance levels for FHB. The results were highly significant and correlated with those obtained using adult plants in the growth chamber and in the field (r = 0.94, P < 0.001 and r = 0.65, P < 0.001, respectively). The Petri-dish test was further investigated for its repeatability and stability in different durum wheat cultivars and highly significant correlation coefficients were obtained (r = 0.90–0.91 (P < 0.001), 0.89–0.95 (P < 0.001), respectively). In this study, we also demonstrated that germination rate reduction and coleoptile length reduction are parameters involved with aggressiveness of F. graminearum. The mean of three disease parameters from the modified Petri-dish method is introduced in this paper as a new parameter for aggressiveness and named “Petri-dish aggressiveness index”. The results obtained reveal that this modified Petri-dish test is rapid, reliable and stable with different durum wheat cultivars, and yields highly significant correlation coefficients with floret and ear inoculations, thus it is suitable to be used for quantification of aggressiveness of F. graminearum.  相似文献   

15.
This study evaluated the impact of time between the application of cell suspensions or cell-free filtrates of Bacillus subtilis strains SB01 or SB24 on soybean plants under field conditions and inoculation with Sclerotinia sclerotiorum on their effectiveness for suppression of S. sclerotiorum. The results showed that the cell suspensions of two strains provided greater effectiveness than the cell-free filtrates, but the suppression effectiveness decreased as the time between application in the field and S. sclerotiorum inoculation increased. The B. subtilis cell suspensions applied on soybean leaves for up to 10 days under field conditions were able to provide a significant (P < 0.01) reduction in disease severity by approximately 20–90% at 5 days after the S. sclerotiorum inoculation. When rated 15 days after S. sclerotiorum inoculation, plants treated with bacterial cells for ≤6 days reduced Sclerotinia stem rot severity by 15–70%. Most effectiveness was provided by the cell suspensions present on soybean leaves for <3 days under field conditions, which significantly (P < 0.01) reduced disease severity by 40–70% over 15 days. In comparison, the cell-free filtrates remaining on leaves for <6 days significantly (P < 0.01) reduced disease severity during the first 5 days after the inoculation, while the best cell-free filtrate treatments were those with ≤1-day intervals, which significantly (P < 0.01) reduced disease severity by 10–40% during 15 days after the inoculation. The effectiveness of B. subtilis was reduced when it rained after application.  相似文献   

16.
Sclerotinia stem rot caused by Sclerotinia sclerotiorum is a serious threat to oilseed production in Australia. Eight isolates of S. sclerotiorum were collected from Mount Barker and Walkway regions of Western Australia in 2004. Comparisons of colony characteristics on potato dextrose agar (PDA) as well as pathogenicity studies of these isolates were conducted on selected genotypes of Brassica napus and B. juncea. Three darkly-pigmented isolates (WW-1, WW-2 and WW-4) were identified and this is the first report of the occurrence of such isolates in Australia. There was, however, no correlation between pigmentation or colony diameter on PDA with the pathogenicity of different isolates of this pathogen as measured by diameter of cotyledon lesion on the host genotypes. Significant differences were observed between different isolates (P ≤ 0.001) in two separate experiments in relation to pathogenicity. Differences were also observed between the different Brassica genotypes (P ≤ 0.001) in their responses to different isolates of S. sclerotiorum and there was also a significant host × pathogen interaction (P ≤ 0.001) in both experiments. Responses between the two experiments were significantly correlated in relation to diameter of cotyledon lesions caused by selected isolates (r = 0.79; P < 0.001, n = 48). Responses of some genotypes (e.g., cv. Charlton) were relatively consistent irrespective of the isolates of the pathogen tested, whereas highly variable responses were observed in some other genotypes (e.g., Zhongyou-ang No. 4, Purler) against the same isolates. Results indicate that, ideally, more than one S. sclerotiorum isolate should be included in any screening programme to identify host resistance. Unique genotypes which show relatively consistent resistant reactions (e.g., cv. Charlton) across different isolates are the best for commercial exploitation of this resistance in oilseed Brassica breeding programmes.  相似文献   

17.
A conventional PCR and a SYBR Green real-time PCR assays for the detection and quantification of Phytophthora cryptogea, an economically important pathogen, have been developed and tested. A conventional primer set (Cryp1 and Cryp2) was designed from the Ypt1 gene of P. cryptogea. A 369 bp product was amplified on DNA from 17 isolates of P. cryptogea. No product was amplified on DNA from 34 other Phytophthora spp., water moulds, true fungi and bacteria. In addition, Cryp1/Cryp2 primers were successfully adapted to real-time PCR. The conventional PCR and real-time PCR assays were compared. The PCR was able to detect the pathogen on naturally infected gerbera plants and on symptomatic artificially infected plants collected 21 days after pathogen inoculation. The detection limit was 5 × 103 P. cryptogea zoospores and 16 fg of DNA. Real-time PCR showed a detection limit 100 times lower (50 zoospores, 160 ag of DNA) and the possibility of detecting the pathogen in symptomless artificially infected plants and in the re-circulating nutrient solution of closed soilless cultivation systems.  相似文献   

18.
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   

19.
Nematode reproduction on the nematode-susceptible tomato cv. Durinta grafted onto the Mi-resistance gene tomato rootstock SC 6301 was compared to the Mi-resistance gene tomato cv. Monika in a plastic house infested with Meloidogyne javanica. The ungrafted susceptible cv. Durinta was included as a control for reference. Final soil population densities were lower (P ≤ 0.05) on the resistant than susceptible cultivar but intermediate values were recorded on the rootstock SC 6301. The lowest numbers of eggs per gram root were recorded on the resistant cultivar followed by those on the rootstock; in both cases, they were lower (P < 0.05) than on the susceptible control. Cumulative yield (kilogram per square meter) was higher (P < 0.05) on the resistant than susceptible cultivar whether or not it had been grafted. The rootstock SC 6301 provided an intermediate resistance response to M. javanica and was less effective than the resistant cultivar in suppressing nematode populations and plant damage under the experimental conditions of this study.  相似文献   

20.
Laboratory studies assessing the degree of suitability of the mealybugs Planococcus ficus (Signoret), Planococcus citri (Risso) and Pseudococcus calceolariae (Maskell), towards the Sicilian ecotype of the encyrtid parasitoid Anagyrus sp. nr. pseudococci were carried out. All three species of mealybugs were shown to encapsulate the eggs of the encyrtid; however, significant differences were revealed among them in rates of encapsulation and/or superparasitism. The level of aggregate encapsulation and effective encapsulation of the parasitoid eggs by P. citri (74.95 ± 0.87 and 60.19 ± 1.70, respectively) was significantly higher than that recorded for P. ficus (aggregate encapsulation = 58.43 ± 0.83 and effective encapsulation = 31.31 ± 1.55) and significantly lower than the values found in Ps. calceolariae (93.99 ± 0.97 and 88.61 ± 2.03, respectively). As such, rates of parasitism were significantly lower for P. ficus compared with both P. citri and Ps. calceolariae. The two latter species of mealybugs showed similar rates of parasitism by A. sp. nr. pseudococci. Moreover, a significantly higher rate of superparasitism was found for Ps. calceolariae compared with both P. citri and P. ficus, whereas no significant differences were found between the two species of Planococcus for this parameter.  相似文献   

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