Selection,characterization and genetic analysis of laboratory mutants of <Emphasis Type="Italic">Botryotinia fuckeliana</Emphasis> (<Emphasis Type="Italic">Botrytis cinerea</Emphasis>) resistant to the fungicide boscalid

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.     

Effects of some fungicides on <Emphasis Type="Italic">Isaria farinosa</Emphasis>, and <Emphasis Type="Italic">in vitro</Emphasis> growth and infection rate on <Emphasis Type="Italic">Planococcus citri</Emphasis>

The effects of some fungicides used against citrus diseases, on mycelial growth and conidial germination of Isaria farinosa (Holmsk.) Fries [Sordariomycetes: Hypocreales] and also on the pathogenicity of the fungus on citrus mealybug, Planococcus citri (Risso), were determined. Systemic fungicides such as tebuconazole, penconazole and nuarimol were the most effective as regards both conidial germination and mycelial growth. Protective fungicides such as captan, chlorothalonil, mancozeb and propineb inhibited conidial germination at between 1 and 5 μg ml⁻¹ concentration, but captan, chlorothalonil and propineb did not inhibit the mycelial growth at 5,000 μg ml⁻¹. Mancozeb inhibited mycelial growth between 2,500 and 5,000 μg ml⁻¹. Sulphur and copper oxychloride did not inhibit the fungus even at very high concentrations. Sulphur, copper oxychloride, fosetyl-al, chlorothalonil and carbendazim did not decrease the mortality percentage caused by I. farinosa. Tebuconazole, penconazole and mancozeb were the most effective and respectively reduced the mortality from 83% to 33%, 28% and 30% in the ovisacs, from 81% to 29%, 27% and 29% in the 1st instar larvae, and from 84% to 34% in the adult females.     

A molecular mechanism of resistance to streptomycin in <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzicola</Emphasis>

Xanthomonas oryzae pv. oryzicola, the causal agent of rice leaf streak disease, was found to be sensitive to streptomycin (an aminocyclitol glycoside antibiotic), by inhibition of protein synthesis resulting from interference with translational proofreading. This study aimed to determine the molecular resistance mechanism of X. oryzae pv. oryzicola to streptomycin. Seven streptomycin-resistant mutants were obtained by UV induction or streptomycin selection. These mutants can grow at 100 μg ml⁻¹ of streptomycin while the wild-type strain (RS105) cannot grow at 5 μg ml⁻¹. Sequencing indicated that the rpsL gene encoding ribosomal protein S12 has 375 bp encoding 125 amino acid residues. In all resistant strains, a mutation in which AAG was substituted for AGG (Lys→Arg) occurred either at codon 43 or 88. Two plasmids, pUFRRS and pUFRRX, were constructed by ligating the rpsL gene into the cosmid pUFR034. The plasmids pUFRRS and pUFRRX containing the Lys→Arg mutation of the rpsL gene conferred streptomycin resistance to the sensitive wild-type strain by electroporation. Both transformants, RS1 and RS2, could grow in the medium containing 50 μg ml⁻¹ of streptomycin. A mutation at codon 43 or 88 in rpsL can result in resistance of Xanthomonas oryzae pv. oryzicola to streptomycin.     

Sensitivity of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> to propamidine: in vitro determination of baseline sensitivity and the risk of resistance

The baseline sensitivity of Botrytis cinerea to propamidine and assessment of the risk of propamidine resistance in vitro are presented in this article. The baseline sensitivities of 41 wild-type strains were distributed as a unimodal curve with EC₅₀ values of mycelial growth ranging from 0.182 to 1.460 μg ml⁻¹, with a mean of 0.79 ± 0.27 μg ml⁻¹. A total of 10 resistant mutants, obtained from one parental strain, were induced by UV irradiation and selected for resistance to propamidine with an average frequency of 1.98 × 10⁻⁹ and 0.025 respectively. These mutants were divided into three classes of resistant phenotypes with low (LR), moderate (MR) and high (HR) levels of resistance, determined by the EC₅₀ values of 5.0–15.0 μg ml⁻¹, 15.1–75.0 μg ml⁻¹ and more than 75.0 μg ml⁻¹ respectively. Neither positive cross-resistance nor negative cross-resistance was detected between propamidine and the fungicides, benzimidazole carbendazim, anilino-pyrimidine pyrimethanil, dicarboximide iprodione or procymidone. All 10 propamidine-resistant mutants showed reduced mycelial growth in vitro, sporulation, spore germination and pathogenicity when compared with the parental strain. These studies demonstrated that propamidine possesses a low risk of resistance developing. However, as B. cinerea is a high-risk pathogen, appropriate precautions against resistance development should be taken.     

<Emphasis Type="Italic">Paecilomyces lilacinus</Emphasis>, a potential biocontrol agent on apple rust mite <Emphasis Type="Italic">Aculus,schlechtendali</Emphasis> and interactions with some fungicides <Emphasis Type="Italic">in vitro</Emphasis>

The apple rust mite Aculus schlechtendali (Nal.) (Acari: Eriophyidae), is a main pest in apple-growing areas in Ankara, Turkey, and chemical control applications have some limitations. Entomopathogenic fungi have a potential for biological control of mites. In this study, an entomopathogenic fungus, Paecilomyces lilacinus (Thom) Samson (Deuteromycota: Hyphomycetes), was first isolated from the mite cadavers on Japanese crab apple leaves and pathogenicity of the fungus was observed in different inoculum densities and relative humidities. The pathogen caused up to 98.22% mortality of the mite population. The effects of some fungicides on the entomopathogenic fungus were determined in in vitro studies. Carbendazim, penconazole and tebuconazole were the most effective fungicides on mycelial growth of P. lilacinus, with EC₅₀ values under 3 μg ml⁻¹. In spore germination tests, captan, mancozeb, propineb were the most effective fungicides, followed by tebuconazole, penconazole, nuarimol and chlorothalonil. Sulphur could not inhibit the conidia germination totally at 5,000 μg ml⁻¹. Copper oxychloride and fosetyl-al prevented conidia formation at concentrations above 1,000 μg ml⁻¹.     

Evaluation of nematicidal properties of saponins from <Emphasis Type="Italic">Medicago</Emphasis> spp.

The nematicidal activity of saponins from Medicago arborea (tops), M. arabica (tops and roots) and M. sativa (tops and roots) against the plant-parasitic nematode Xiphinema index was investigated. Nematicidal activity of related prosapogenins and sapogenins on X. index is also described. Saponins from Medicago spp. at different concentrations were all nematicidal, those from M. arborea tops being the less effective. In general, saponins induced 100% mortality at 500 μg ml⁻¹ between 8 and 48 h, while prosapogenins resulted in toxicity starting at 125 μg ml⁻¹. Differences in the effects on X. index induced by prosapogenins and sapogenins were less pronounced, although prosapogenins displayed a larger range of activity. Assays with purified sapogenins demonstrated the relationship of the observed nematicidal activity of M. sativa and M. arborea to the content of the main aglycones (medicagenic acid and hederagenin, respectively) in the saponin extracts. Hederagenin displayed the highest bioactivity, giving 38% mortality after 1 h at 125 μg ml⁻¹.     

Pathogenicity of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> against <Emphasis Type="Italic">Galleria mellonella</Emphasis>

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.     

Potential inhibitors against <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>, produced by the fungus <Emphasis Type="Italic">Myrothecium</Emphasis> sp. associated with the marine sponge <Emphasis Type="Italic">Axinella</Emphasis> sp.

Sclerotinia sclerotiorum is a worldwide ascomycete fungal plant pathogen, which causes enormous yield losses on major economic crops such as crucifers, grain legumes and several other plant families. The objective of this research was to isolate and characterise some bioactive products from cultures of fungi associated with the marine sponge Axinella sp. In total, nine fungal isolates were obtained from the marine sponge Axinella sp. collected from the South China Sea. A group of test strains, including two G⁺ strains (Bacillus subtilis and Staphylococcus aureus), two G⁻ strains (Escherichia coli and Pseudomonas aeruginosa) and three fungi including two plant pathogenic fungi Sclerotinia sclerotiorum and Magnaporthe grisea and Saccharomyces cerevisiae, were employed as the indicator organisms for bioactivity screening. Using antagonistic tests and bioactive screening of the ethyl acetate (EtOAc) extracts of the corresponding cultures, fungal isolate JS9 showed the stronger efficacy against the test indicator strains, especially the indicator fungal pathogens. Isolate JS9 was further identified as Myrothecium sp. by a combination of morphological features and 18S rDNA BLAST on GenBank. Two macrocyclic trichothecenes, roridin A (compound 1) and roridin D (compound 2) were purified by tracking the activity of the EtOAc extract fractions and characterised with spectral analyses including MS, ¹H-NMR, ¹³C-NMR and disortionless enhancement by polarization transfer (DEPT). In vitro antifungal tests showed that the two macrocyclic trichothecenes were bioactive against S. cerevisiae, M. grisea and S. sclerotiorum with minimal inhibitory concentrations of 31.25, 125 and 31.25 μg ml⁻¹ for roridin A, and 62.5, 250 and 31.25 μg ml⁻¹ for roridin D, respectively. The present investigation demonstrated that two antifungal trichothecenes including roridin A and roridin D produced by the fungus Myrothecium sp. isolated from the marine sponge Axinella sp. could be potential inhibitors against the plant pathogen S. sclerotiorum. Lian Wu Xie and Shu Mei Jiang contributed equally to this work.     

Susceptibility of eggs of <Emphasis Type="Italic">Tribolium confusum</Emphasis>, <Emphasis Type="Italic">Ephestia kuehniella</Emphasis> and <Emphasis Type="Italic">Plodia interpunctella</Emphasis> to four essential oil vapors

Susceptibility of eggs of Tribolium confusum du Val. (Coleoptera: Tenebrionidae), Ephestia kuehniella (Zell.) (Lepidoptera: Phycitidae) and Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae) to vapors of essential oil from garlic (Allium sativum L.), birch (Betula lenta L.), cinnamon (Cinnamonum zeylanicum (Blume)) and aniseed (Pimpinella anisum L.) was studied. Preliminary bioassay tests indicated that vapors of the essential oils had a significant effect on the eggs of tested insect species when exposed to a concentration of 20 μl l ⁻¹ air for 24 h. Generally, garlic and birch essential oils were more toxic to the eggs of tested insect species than cinnamon and aniseed essential oils (except for eggs of T. confusum). There was also a significant difference between susceptibility of eggs of T. confusum, E. kuehniella and P. interpunctella to tested essential oils. Toxicity data indicated that eggs of T. confusum were more susceptible to tested essential oils, with LC₉₀ values ranging from 3.11 to 33.49 μl l ⁻¹ air, than those of E. kuehniella and P. interpunctella; eggs of P. interpunctella were the most tolerant to the essential oils, with LC₉₀ values ranging from 22.02 to 72.42 μl l ⁻¹ air. Concentration × time (Ct) products of 0.29, 0.22, 0.13 and 1.37 mg h l ⁻¹ for garlic, birch, cinnamon and aniseed essential oil, respectively, were required to obtain 90% kill of T. confusum eggs. Although cinnamon essential oil had a much closer Ct product value to methyl bromide, garlic and birch essential oils were found to be the most promising ones since they had also high fumigant toxicity on eggs of both E. kuehniella and P. interpunctella.     

The activity of amino acids produced by <Emphasis Type="Italic">Paenibacillus macerans</Emphasis> and from commercial sources against the root-knot nematode <Emphasis Type="Italic">Meloidogyne exigua</Emphasis>

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.     

Detection of viridiofungin A and other antifungal metabolites excreted by <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> active against different plant pathogens

Antibiosis is assumed to be an essential mechanism exerted by potential biocontrol agents (BCAs) of Trichoderma spp. Therefore, in the present study, we report for the first time on the elucidation and production of viridiofungin A (VFA) from T. harzianum isolate T23 cultures and investigate the antifungal potential of VFA and some other secondary metabolites purified from T. harzianum cultures against Fusarium moniliforme. The bioautography assay revealed that T. harzianum isolates T16 and T23 excreted several secondary metabolites with antifungal activity. Following isolation and purification of the antifungal zones, three fractions (F223, F323 and F423) from extracts of isolate T23 and two fractions (F416 and F516) from extracts of isolate T16 exhibited pronounced fungitoxic activity in the bioautography and antibiotic disk assays against Cladosporium spp. and F. moniliforme, respectively. The structure of the antifungal metabolite in fraction F323 was identified as viridiofungin A (VFA), the first report of production of VFA by isolate T23 of T. harzianum. Following cultivation of isolate T23 in PDB medium for 9 days, 94.6 mg l⁻¹ of VFA were determined. VFA and fraction F516 retarded the mycelial growth of F. moniliforme in the non-volatile phase assay by >90% for each 250 μg ml⁻¹ 7 days post-inoculation (dpi). While VFA and fraction F416 showed both volatile and non-volatile effects, fraction F516 seemed to exhibit mainly non-volatile activity. Microscopic examination revealed that hyphae of F. moniliforme grown on VFA-amended medium were less branched and appeared thicker than untreated hyphae. Furthermore, in the presence of VFA, formation of chlamydospores by F. moniliforme was increased. Finally, the antifungal spectrum of VFA towards various important plant pathogens was evaluated. Germination of propagules of a variety of fungal pathogens in vitro was differentially inhibited by VFA. While in the presence of 100 μg ml⁻¹ VFA conidial germination of V. dahliae was completely inhibited, a slightly higher concentration (150 μg ml⁻¹) of the inhibitor was required to suppress germination of Phytophthora infestans sporangia or sclerotia of Sclerotinia sclerotiorum. Contrary to several reports in the literature, VFA proved to be fungistatic rather than fungicidal. However, neither VFA nor the other Trichoderma metabolites, such as 6PAP, F416 and F516, exhibited any antibacterial activity against Gram-positive and Gram-negative bacteria.     

Effect of seed priming with <Emphasis Type="Italic">Serratia plymuthica</Emphasis> and <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> to control <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> in different oilseed rape cultivars

The efficacy of a seed treatment of oilseed rape (OSR) (Brassica napus) with the rhizobacteria Serratia plymuthica (strain HRO-C48) and Pseudomonas chlororaphis (strain MA 342) applied alone or in combination against the blackleg disease caused by Leptosphaeria maculans was tested with different cultivars. Seeds were soaked in bacterial suspensions (bio-priming) to obtain log₁₀ 6–7 CFU seed⁻¹. Cotyledons were inoculated with a 10 ul droplet of L. maculans spore suspension of log₁₀ 7 spores ml⁻¹ and the disease index (size of lesions) was evaluated 14 days later. A mean disease reduction of 71.6% was recorded for S. plymuthica and of 54% for P. chlororaphis. The combined treatment was not superior to the treatment with S. plymuthica alone. The reduction of the disease caused by S. plymuthica was independent of the cultivar’s susceptibility, whereas the control effect recorded with P. chlororaphis increased with decreasing cultivar resistance to blackleg disease. The bacterial colonization of OSR was restricted to the roots and hypocotyl. No significant difference in bacterial colonization of the rhizosphere was observed between different cultivars, nor between single or combined bacterial seed treatments.     

High Performance Thin Layer Chromatography (HPTLC) profile of an insecticidal compound of <Emphasis Type="Italic">Trochus radiatus</Emphasis> and assessment of mucus-associated bacterial extracts

The current study is based on the screening of novel insecticides from new sources that have not being exploited hitherto. The major objectives of this research work were to extract marine molluscs, Lambis lambis, Trochus radiatus and Chicoreus ramosus, from Tuticorin coastal waters using different solvents to test their insecticidal properties and partially purify the active components. The ethyl acetate extracts of L. lambis and T. radiatus showed 100% mortality of Sitophilus oryzae, at a concentration of 200 μg ml⁻¹. The LC₅₀ values for ethyl acetate extracts of L. lambis, T. radiatus and C. ramosus were found to be 67.08, 348.86 and 571.42 μg ml⁻¹, respectively. With regard to bacterial metabolites, at a concentration of 200 μg ml⁻¹ the ethyl acetate extract of A3 was able to elicit an activity of 40% and that of strain A1 – 20%. The LC₅₀ values of the bacterial metabolites were also investigated. Upon chromatographic separation of active ethyl acetate extracts of T. radiatus, the 100% methanol column-purified fraction was found to have an activity of 30% at a concentration of 10 μg against S. oryzae. The purity of the partially pure active compound was observed to be 72.78% on analysis with high pressure thin layer chromatography.     

Note: Evaluation of antibacterial activity of essential oil of<Emphasis Type="Italic">Rosa damascena</Emphasis> on<Emphasis Type="Italic">Erwinia amylovora</Emphasis>

Significant antibacterial activity was observed in the essential oil (E.O.) ofRosa damascena Mill. and the Minimum Bactericidal Concentration (MBC) of the E.O. was determined as 1386.5 μg ml⁻¹ forErwinia amylovora, the causal agent of fire blight disease. Ooze formation on immature pears and lesion formation in artificially inoculated shoots were completely (100%) prevented by the essential oil ofR. damascena (1500 μg ml⁻¹), essential oil ofThymbra spicata var.spicata (500 μg ml⁻¹) and streptomycin (100 μg ml⁻¹). Copper oxychloride plus maneb significantly reduced ooze formation and lesion formation, but the control was less than that obtained with the essential oils or streptomycin. The essential oil ofR. damascena may be a useful natural bactericide for the control of the fire blight pathogen,E. amylovora. http://www.phytoparasitica.org posting July 14, 2004.     

Effect of indole-acetic acid (IAA) on the development of symptoms caused by <Emphasis Type="Italic">Pythium ultimum</Emphasis> on tomato plants

The effect of indole-acetic acid (IAA) on the development of symptoms caused by Pythium ultimum on tomato plants was investigated using different bioassays. Application of IAA (5 μg ml⁻¹) on tomato seedlings inoculated with P. ultimum did not affect their emergence suggesting that IAA did not affect the severity of Pythium damping-off. However, IAA was shown to influence the development of P. ultimum symptoms on tomato plantlets. Low concentrations of IAA (0–0.1 μg ml⁻¹) within the rhizosphere of plantlets increased the severity of the symptoms caused by P. ultimum, while higher concentrations (10 μg ml⁻¹), applied either by drenching to the growing medium or by spraying on the shoot, reduced the symptoms caused by this pathogen. In addition, the study demonstrated that P. ultimum produces IAA in liquid culture amended with L-tryptophan, tryptamine or tryptophol (200 μg ml⁻¹) and in unamended culture.     

Toxigenicity and pathogenicity of <Emphasis Type="Italic">Fusarium poae</Emphasis> and <Emphasis Type="Italic">Fusarium avenaceum</Emphasis> on wheat

In a field experiment between 2004 and 2006, 14 winter wheat varieties were inoculated with either a mixture of three isolates of F. poae or a mixture of three isolates of F. avenaceum. In a subsequent climate chamber experiment, the wheat variety Apogee was inoculated with individual single conidium isolates derived from the original poly conidium isolates used in the field. Disease symptoms on wheat heads were visually assessed, and the yield as well as the fungal incidence on harvested grains (field only) was determined. Furthermore, grains were analysed using LC-MS/MS to determine the content of Fusarium mycotoxins. In samples from field and climate chamber experiments, 60 to 4,860 μg kg⁻¹ nivalenol and 2,400 to 17,000 μg kg⁻¹ moniliformin were detected in grains infected with F. poae and F. avenaceum, respectively. Overall, isolate mixtures and individual isolates of F. avenaceum proved to be more pathogenic than those of F. poae, leading to a higher disease level, yield reductions up to 25%, and greater toxin contamination. For F. poae, all variables except for yield were strongly influenced by variety (field) and by isolate (climate chamber). For F. avenaceum, variety had a strong effect on all variables, but isolate effects on visual disease were not reflected in toxin production. Correlations between visual symptoms, fungal incidence, and toxin accumulation in grains are discussed.     

Pathogenicity and mycotoxin production by <Emphasis Type="Italic">Fusarium proliferatum</Emphasis> isolated from onion and garlic in Serbia

Fusarium proliferatum can occur on a wide range of economically important vegetable plants but its role in disease is not always well established. In 2000 and 2001, from forty-one field samples of wilting onion and garlic plants in Serbia, F. proliferatum as the predominant fungal species was isolated from root and bulbs. Seventy isolates were firstly characterized for their sexual fertility and were shown to be mostly members of Gibberella intermedia (sixty-seven of seventy isolates, the remaining three isolates were unfertile), the sexual stage of F. proliferatum (syn. mating population D of G. fujikuroi complex). A selected set of eleven F. proliferatum isolates from both hosts were also tested for their pathogenicity and toxigenicity. Although onion and garlic plants were susceptible to all isolates, onion plants showed a significantly higher disease severity index. Six of the eleven isolates of F. proliferatum produced fumonisin B₁ from 25 to 3000 μg g⁻¹, and beauvericin from 400 to 550 μg g⁻¹; ten isolates produced fusaric acid from 80 to 950 μg g⁻¹ and moniliformin from 50 to 520 μg g⁻¹. Finally, all isolates produced fusaproliferin up to 400 μg g⁻¹. These results confirm F. proliferatum as an important pathogen of garlic and onion in Europe and that there is a potential mycotoxin accumulation risk in contaminated plants of both garlic and onion.     

EMS and UV irradiation induce unstable resistance against CAA fungicides in <Emphasis Type="Italic">Bremia lactucae</Emphasis>

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.     

Effect of botanical extracts on the biological activity of granulosis virus against <Emphasis Type="Italic">Pieris brassicae</Emphasis>

A granulosis virus strain infecting Pieris brassicae (PbGV) was isolated from the dry temperate region of northwestern Himalayas as a potential microbial agent for its management. The effect of different botanicals (having insecticidal action against P. brassicae) on the bioefficacy of PbGV was evaluated under laboratory conditions using leaf disc bioassays on cabbage for improving the insecticidal performance of the PbGV. The synergistic action of different botanical extracts was evident in terms of reduction in LC₅₀ values against different botanical extracts. Among different extracts, petroleum-ether extract of neem seed kernel (NSK) when combined with PbGV resulted in maximum reduction of LC₅₀ value (4.39 × 10² occlusion bodies [OBs] ml⁻¹) followed by methanolic extract (7.38 × 10² OBs ml⁻¹) and aqueous extract (9.36 × 10³ OBs ml⁻¹) as compared with PbGV alone (1.85 × 10⁴ OBs ml⁻¹) for 2nd instar larvae of the test insect. These trends were found analogous in cases of 3rd and 4th instars of P. brassicae with different solvent extracts of NSK. The other botanicals evaluated, viz., Eupatorium and Artemesia, also resulted in reduction of LC₅₀ values for 2nd, 3rd and 4th instars as compared with PbGV alone when different extracts were combined with virus for bioassays. The studies suggest that the PbGV in combination with botanical pesticides could be more useful as a bio-pesticide against cabbage butterfly (P. brassicae) in IPM programs.     

Aggressiveness and mycotoxin production of eight isolates each of <Emphasis Type="Italic">Fusarium graminearum</Emphasis> and <Emphasis Type="Italic">Fusarium verticillioides</Emphasis> for ear rot on susceptible and resistant early maize inbred lines

Fusarium graminearum and F. verticillioides are among the most important pathogens causing ear rot of maize in Central Europe. Our objectives were to (1) compare eight isolates of each species on two susceptible inbred lines for their variation in ear rot rating and mycotoxin production across 3 years, and (2) analyse two susceptible and three resistant inbred lines for potential isolate x line interactions across 2 years by silk-channel inoculation. Ear rot rating, zearalenone (ZEA) and deoxynivalenol (DON) concentrations were evaluated for all F. graminearum isolates. In addition, nivalenol (NIV) concentrations were analysed for two NIV producers. Fumonisin (FUM) concentrations were measured for all F. verticillioides isolates. Mean ear rot severity was highest for DON producers of F. graminearum (62.9% of the ear covered by mycelium), followed by NIV producers of the same species (24.2%) and lowest for F. verticillioides isolates (9.8%). For the latter species, ear rot severities differed highly among years (2006: 24%, 2007: 3%, 2008: 7%). Mycotoxin concentrations among isolates showed a broad range (DON: 100–284 mg kg⁻¹, NIV: 15–38 mg kg⁻¹, ZEA: 1.1–49.5 mg kg⁻¹, FUM: 14.5–57.5 mg kg⁻¹). Genotypic variances were significant for isolates and inbred lines in all traits and for both species. Isolate x line interactions were significant only for ear rot rating (P < 0.01) and DON concentration (P < 0.05) of the F. graminearum isolates, but no rank reversals occurred. Most isolates were capable of differentiating the susceptible from the resistant lines for ear rot severity. For resistance screening, a sufficiently aggressive isolate should be used to warrant maximal differentiation among inbred lines. With respect to F. verticillioides infections, high FUM concentrations were found in grains from ears with minimal disease symptoms.