Impact of culture age on conidial germination,desiccation and UV tolerance of entomopathogenic fungi

The impact of culture age on conidial yields, germination and tolerance to UV exposure of freshly harvested and dry conidia produced by five entomopathogenic fungal (EPF) isolates was studied. Beauveria bassiana, Metarhizium anisopliae, Lecanicillium lecanii and Lecanicillium muscarium were grown on potato dextrose agar (PDA) medium for 7 or 14 days at 25°C. While the age of cultures had a significant impact on the germination rate of conidia produced by isolates L. lecanii CBS 122.175 and B. bassiana LMSA 1.01.093, other EPF isolates germinated at the same rate regardless of the culture age. When exposed to UV radiation, conidia produced by all isolates germinated at a lower rate compared to the non-irradiated conidia, although this decrease in germination (20–80% decrease) was unaffected by the culture age. Air-drying had only a slight impact on conidial germination (0–60% decrease). Under the conditions of this study, the stability of irradiated conidia produced by M. anisopliae LMSA 1.01.197 and B. bassiana CBS 110.25 was significantly increased when conidia were dried prior to UV exposure. This increase in tolerance to stress of dried conidia might be caused, at least partially, by the low metabolic activity associated with dehydration.     

Suppression of Ripe Rot on ‘Zesy002’ Kiwifruit with Commercial Agrochemicals

Ripe rot caused by Botryosphaeria dothidea is one of the serious diseases of postharvest kiwifruit. In order to control ripe rot on Actinidia chinensis cultivar ‘Zesy002’, several commercial agrofungicides were selected by an antifungal test on an artificial medium. Furthermore, disease suppression by the selected fungicides was evaluated on the kiwifruit by inoculation with a conidial suspension of B. dothidea. On the artificial media containing boscalid + fludioxonil was shown to be the most effective antifungal activity. However, in the bio-test pyraclostrobin + boscalid and iminoctadine-tris were the most effective agrochemicals on the fruit. On the other hand, the infection structures of B. dothidea on kiwifruit treated with pyraclostrobin + boscalid were observed with a fluorescent microscope. Most of the fungal conidia had not germinated on the kiwifruit treated with the agrochemicals whereas on the untreated fruit the fungal conidia had mostly germinated. Electron microscopy of the fine structures showed morphological changes to the conidia and branch of hyphae on the kiwifruit pre-treated with pyraclostrobin + boscalid, indicating its suppression effect on fungal growth. Based on this observation, it is suggested that ripe rot by B. dothidea may be suppressed through the inhibition of conidial germination on the kiwifruit treated with the agrochemicals.     

Pathogenicity of entomopathogenic fungi to eggs and larvae of Spodoptera litura,the common cutworm

The eggs and second instar larvae of Spodoptera litura were treated with different concentrations of conidial suspensions of six isolates of fungi belonging to five species, Metarhizium anisopliae var. anisopliae (Metschnikov) Sorokin (ARSEF 7487), Lecanicillium muscarium (Petch) Zare & W Gams (ARSEF 7037 and ARSEF 6118), Cordyceps cardinalis (ARSEF 7193), Fusarium lateritium Nees (ARSEF 8291/MTCC 9050) and Aspergillus sp. (ARSEF 8519). In bioassay with unscaled eggs, M. anisopliae, C. cardinalis, F. lateritium and Aspergillus sp. resulted in 100% mortality above 10⁶ conidia/mL. However, with scaled egg masses, the highest hatching rate (56%) was observed with L. muscarium (ARSEF 6118) whereas the lowest hatchings were observed in the case of M. anisopliae followed by L. muscarium (ARSEF 7037), Aspergillus sp., F. lateritium, and C. cardinalis. The larvae were also found susceptible to all isolates in a dose dependent manner. Three promising isolates against larvae, viz. M. anisopliae, F. lateritium and L. muscarium (ARSEF 7037) resulted in average percent mortalities of 88, 89 and 77%, respectively at ≈10⁸ conidia/mL. When both larvae and the leaves (provided as food) were treated with ≈10⁸ conidia/mL, mortality further increased for these isolates. Based on the mortality data and the LC₅₀ values, we suggest that M. anisopliae, F. lateritium and L. muscarium (ARSEF 7037) could be developed as potential biocontrol agents against rice cutworm in IPM programs.     

Efficient germ-line transformation of the economically important pest species Lucilia cuprina and Lucilia sericata (Diptera, Calliphoridae)

The green blowfly species Lucilia cuprina and Lucilia sericata are economically important pests for the sheep industries of Australia and New Zealand. L. cuprina has long been considered a good target for a genetic pest management program. In addition, L. sericata maggots are used in the cleaning of wounds and necrotic tissue of patients suffering from ulcers that are difficult to treat by other methods. Development of efficient transgenesis methods would greatly facilitate the development of strains ideal for genetic control programs or could potentially improve “maggot therapy”. We have previously reported the germ-line transformation of L. cuprina and the design of a “female killing system” that could potentially be applied to this species. However, the efficiency of transformation obtained was low and transformed lines were difficult to detect due to the low expression of the EGFP marker used. Here we describe an efficient and reliable method for germ-line transformation of L. cuprina using new piggyBac vector and helper plasmids containing the strong promoter from the L. cuprina hsp83 gene to drive expression of the transposase and fluorescent protein marker gene. We also report, for the first time, the germ-line transformation of L. sericata using the new piggyBac vector/helper combination.     

Relative susceptibility of <Emphasis Type="Italic">Spodoptera litura</Emphasis> pupae to selected entomopathogenic fungi

The pupae of Spodoptera litura (Fab.), (Lepidoptera: Noctuidae), a polyphagous pest affecting common crops in Indian subcontinent, were treated with different concentrations of conidia of four isolates of entomopathogenic fungi belonging to three species, Metarhizium anisopliae var. anisopliae (Metschnikov) Sorokin (ARSEF 7487), Lecanicillium muscarium (Petch) Zare & W Gams (two isolates ARSEF 7037 and ARSEF 6118) and Cordyceps cardinalis Sung & Spatafora (ARSEF 7193) under laboratory conditions. Suspensions (10⁸/ml) of conidia harvested from Sabouraud dextrose agar yeast extract (SDAY) plates resulted in the highest mortality (85.8%) with M. anisopliae and the lowest mortality (57.3%) with C. cardinalis. The values of LC₅₀ and LC₉₀ suggested that M. anisopliae was the most virulent fungal strain followed by L. muscarium (ARSEF 7037). However, C. cardinalis was the least virulent species among the fungi used in the bioassay. In soil bioassays, drenching the soil with conidial suspensions of ARSEF 7487 and ARSEF 7037 (10⁸ conidia/g of soil) reduced the adult emergence from pupa by 81.3% and 72.5%, respectively, while premixing the sterile soil with conidia killed lesser number of pupae (62.9% by ARSEF 7487 and 54.6% by ARSEF 7037). Our findings suggest that M. anisopliae (ARSEF 7487) and L. muscarium (ARSEF 7037) are potent entomopathogens and could be developed into biocontrol agents against rice cutworm in IPM programs. Handling editor: Helen Roy     

Genetic characterization,pathogenicity and benomyl susceptibility of Lecanicillium fungal isolates from Argentina

The aim of this study was to investigate biological and molecular characteristics of Lecanicillium strains isolated from Hemipteran hosts in Argentina. Morphology‐based taxonomic characterization together with molecular taxonomy based on rRNA operon internal transcribed spacer (ITS), mitochondrial nad1 gene, and nuclear ef1a gene sequences resulted in the assignment of nine out of ten isolates to the Lecanicillium lecanii sensu lato complex. However, whereas several isolates were thus unequivocally characterized as Lecanicillium muscarium or Lecanicillium longisporum, species assignment was not possible for three isolates that might represent a new species within the L. lecanii s.l. complex. We found two group‐I introns on 18S and 28S rRNA gene on only one isolate. Pathogenicity tests were conducted against the peach aphid using conidial suspensions (1 × 10⁷ conidia/ml), and the Kaplan–Meier analysis was performed to evaluate the survival of Myzus persicae. Lecanicillium longisporum CEP 155 and L. muscarium CEP 182 were significantly more pathogenic to M. persicae than all the Lecanicillium isolates causing aphid mortalities >85%. Determination of susceptibility to the benzimidazole fungicide benomyl revealed important differences between Lecanicillium strains. The inhibitory effect of benomyl appeared less pronounced for the L. muscarium fungal isolates than for those belonging to a different taxon. Based in our results, the best candidate strain as microbial biological control agent against M. persicae is L. muscarium CEP 182. However, further research under field conditions in greenhouses should be done in order to confirm the compatibility of entomopathogenic fungi and fungicides within an IPM strategy.     

Screening of high toxic Metarhizium strain against Plutella xylostella and its marking with green fluorescent protein

Entomopathogenic fungus is proposed to be one of the best biocontrol agents against the destructive insect pest Plutella xylostella. In this study, we tested the virulence of 11 Metarhizium strain isolates against P. xylostella using a leaf dipping method, and found one strain, named 609, which had displayed the highest pathogenicity. Bioassay results showed that the accumulated corrected mortality rate was 86.7 % on the eighth day after inoculation with a spore concentration 1 × 10⁸ conidia/mL, and that the time to 50 % lethality was 5.7-day. The strain was identified as Metarhizium anisopliae var. acridum by internal transcribed spacer (ITS) region sequencing. A green fluorescent protein (GFP) marker containing vector, camben-gfp, was constructed and delivered into strain 609 by Agrobacterium tumefaciens-mediated transformation. Six positive clones expressing GFP were selected and tested for toxicity against P. xylostella, all of which displayed the same toxicity as the parental wild type strain. The survival rate of transformant T1 was investigated by monitoring GFP levels at 4-day intervals after inoculation into soil. We found that the concentration of Metarhizium spores decreased sharply from 1 × 10⁷ conidia/g to 1 × 10⁶ conidia/g in the first 5 days after inoculation. The decreasing trend then stabilized and the spore count declined to approximately 1 × 10⁴–10⁵ conidia/g after 1 month. The results of this study indicate that the expression of gfp gene in strain 609 does not alter the virulence capability of Metarhizium. This strain will therefore be useful for the control of P. xylostella and as a tool to study molecular biology properties and monitor colonization of M. anisopliae in the field.     

Agrobacterium tumefaciens-Mediated Transformation of the Causative Agent of Valsa canker of Apple Tree Valsa mali var. mali

Valsa mali var. mali (Vmm), which is the causative agent of Valsa canker of apple tree, causes heavy damage to apple production in eastern Asia. In this article, we report Agrobacterium tumefaciens-mediated transformation (ATMT) of Vmm and expression of gfp (green fluorescent protein) in this fungus. The transformation system was optimized to a transformation efficiency of approximately 150 transformants/10⁶ conidia, and a library containing over 4,000 transformants was generated. The tested transformants were mitotically stable. One hundred percent hph (hygromycin B phosphotransferase) integration into Vmm was identified by PCR and five single-copy integration of T-DNA was detected in the eighteen transformants by Southern blot. To our knowledge, this is the first report of ATMT of Vmm. Furthermore, this library has been used to identify genes involved in the virulence of the pathogen, and the transformation system may also be useful to the transformation of other species of the genus Valsa.     

Metarhizium robertsii illuminated during mycelial growth produces conidia with increased germination speed and virulence

Light conditions during fungal growth are well known to cause several physiological adaptations in the conidia produced. In this study, conidia of the entomopathogenic fungi Metarhizium robertsii were produced on: 1) potato dextrose agar (PDA) medium in the dark; 2) PDA medium under white light (4.98 W m^?2); 3) PDA medium under blue light (4.8 W m^?2); 4) PDA medium under red light (2.8 W m^?2); and 5) minimum medium (Czapek medium without sucrose) supplemented with 3 % lactose (MML) in the dark. The conidial production, the speed of conidial germination, and the virulence to the insect Tenebrio molitor (Coleoptera: Tenebrionidae) were evaluated. Conidia produced on MML or PDA medium under white or blue light germinated faster than conidia produced on PDA medium in the dark. Conidia produced under red light germinated slower than conidia produced on PDA medium in the dark. Conidia produced on MML were the most virulent, followed by conidia produced on PDA medium under white light. The fungus grown under blue light produced more conidia than the fungus grown in the dark. The quantity of conidia produced for the fungus grown in the dark, under white, and red light was similar. The MML afforded the least conidial production. In conclusion, white light produced conidia that germinated faster and killed the insects faster; in addition, blue light afforded the highest conidial production.     

Effect of fungicides on growth,germination and cuticle-degrading enzyme production by Lecanicillium muscarium

Lecanicillium muscarium Zare and Gams (previously known as Verticillium lecanii) is a well-known pathogen of arthropods. The influence of six fungicides on growth, sporulation, conidial germination and cuticle-degrading enzyme production by L. muscarium was investigated under laboratory conditions. The maximum reduction in vegetative growth, sporulation and conidial germination in relation to the control treatment was observed for propiconazole, whereas mancozeb and chlorothalonil caused the lowest reduction in these parameters. Propiconazole and pyraclostrobin caused higher reduction in enzyme activities (chitinase, Pr1, Pr2 and lipase) at all three concentrations (10, 100 and 1000 µg/ml), whereas low reduction in enzyme activities was caused by chlorothalonil and mancozeb when used at 10 µg/ml. The data presented can be used for future recommendations of these fungicides in Integrated Pest Management (IPM) programmes where L. muscarium is an important control agent.     

Construction of an RNAi expression vector and transformation into Penicillium chrysogenum

In this work, we constructed an RNAi vector for attenuation of the class III chitin synthase gene chs4, which plays a major role in hyphal growth and conidia formation. To achieve a high transformation frequency, factors affecting the preparation and regeneration of protoplasts were analyzed. The maximum numbers of protoplasts (1.41?×?10⁷ mL^?1) were released when mycelia cultured for 48 h were incubated at 30 °C for 5 h in a buffer containing 4 mg mL^?1 lysing enzyme. The maximum regeneration rate (33 %) was obtained when mycelia were digested for 4 h and plated on a regeneration medium containing 1 % overlaid agar. Quantitative real-time PCR was performed to validate the transformation efficiency, and it revealed knockdown of chs4 gene in randomly selected transformants at different levels. Dramatic reductions in the formation of conidia and the hyphal growth rate were observed in most of the transformants.     

Competition is the mechanism of biocontrol of brown rot in stone fruit by <Emphasis Type="Italic">Penicillium frequentans</Emphasis>

The mechanism of biocontrol of brown rot in stone fruit by Penicillium frequentans Westling (Pf909) was investigated using in vitro and in vivo growth assays and a benomyl-resistant strain of Monilinia fructicola (G Winter) Honey (Mf3C). For the in vitro assays, Pf909 and Mf3C conidia were suspended in Czapek-Dox broth, which was amended or not amended with a skin extract of mature peaches. The growth and germination of Pf909 and Mf3C conidia were determined by counting the number of colony-forming units on potato dextrose agar plates, which were amended or not amended with 0.5 g ml^?1 benomyl. In some of the assays, germinated Pf909 conidia were used before their exposure to Mf3C conidia. For the in vivo assays, healthy cherries were inoculated with Mf3C conidia before and after applying Pf909 conidia on the cherry surface and the incidence of brown rot was recorded for seven days. Since we found that Pf909 conidia compete with Mf3C conidia for space and nutrients in the different assays, we concluded that competition is the probable primary mechanism of biocontrol of Pf909.     

Compatibility among entomopathogenic hyphocreales and two beneficial insects used to control Trialeurodes vaporariorum (Hemiptera: Aleurodidae) in Mediterranean greenhouses

The effect of the combined use of Encarsia formosa or Macrolophus caliginosus and one of three marketed mycoinsecticides, Mycotal® (Leucanicillium muscarium-based), Naturalis-L™ (Beauveria bassiana-based) and PreFeRal® (Isaria fumosorosea-based), on the control of the whitefly, Trialeurodes vaporariorum, was studied under laboratory and greenhouse conditions. The results of both types of tests, the bioassays and the greenhouse trials, for all combinations of E. Formosa with each of the three mycoinsecticides showed that the total mortality of larval populations of T. vaporariorum was not affected. The mortality of T. vaporariorum larvae treated in the second instar revealed the capacity for both B. bassiana- and L. muscarium-based formulations and E. formosa to kill the host either separately or in association. Because of its higher pathogenic activity (under our test conditions), L. muscarium provoked a large proportion of mycoses in larvae exposed to parasitization. In contrast, the efficacy of parasitization was higher in larvae treated with B. bassiana and exposed to E. formosa because of a lower pathogenic activity of the fungus. Bioassays carried out with third-instar larvae of T. vaporariorum showed a low susceptibility to both tested fungi. Consequently, mortalities recorded in larvae subjected to the combined treatments by consecutive exposures or at 2-4 days post-parasitization were mainly caused by the development of the parasitoid. Greenhouse trials showed that fungus-induced mortality of T. vaporariorum in plants treated with L. muscarium, I. fumosorosea, and B. Bassiana was significant compare to control. L. muscarium, B. bassiana and I. fumosorosea killed young whitefly larvae and limited parasitization to 10% or less. Second-instar larvae of M. caliginosus were not susceptible to L. muscarium and B. bassiana formulations with any mode of contamination: direct spraying of larvae, spraying on the foliar substrate or by contaminated T. vaporariorum prey. In greenhouse trials, M. caliginosus populations treated with fungi were not significantly affected compared to controls.     

Germination polarity of Beauveria bassiana conidia and its possible correlation with virulence

Three different germination types of conidia; unidirectional, bidirectional and multidirectional, were revealed through microscopic observations for eight Beauveria bassiana isolates germinated on Sabouraud dextrose agar. Canonical correlation analysis indicated that there is a positive correlation between unidirectional germination and virulence against diamondback moth, Plutella xylostella and the Colorado potato beetle, Leptinotarsa decemlineata. Scanning electron microscopy revealed different in vivo behaviors for unipolar- and bipolar-germinated conidia. Unipolar-germinated conidia produced a strong germ tube with mostly appressorium-like structures while bipolar-germinated conidia continued to invasive hyphal growth without any penetration, indicating that germination polarity in one way or another may be an indicator of pathogenic ability.     

An efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation method for aflatoxin generation fungus <Emphasis Type="Italic">Aspergillus flavus</Emphasis>

Aspergillus flavus often invade many important corps and produce harmful aflatoxins both in preharvest and during storage stages. The regulation mechanism of aflatoxin biosynthesis in this fungus has not been well explored mainly due to the lack of an efficient transformation method for constructing a genome-wide gene mutant library. This challenge was resolved in this study, where a reliable and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for A. flavus NRRL 3357 was established. The results showed that removal of multinucleate conidia, to collect a homogenous sample of uninucleate conidia for use as the transformation material, is the key step in this procedure. A. tumefaciens strain AGL-1 harboring the ble gene for zeocin resistance under the control of the gpdA promoter from A. nidulans is suitable for genetic transformation of this fungus. We successfully generated A. flavus transformants with an efficiency of ～ 60 positive transformants per 10⁶ conidia using our protocol. A small-scale insertional mutant library (～ 1,000 mutants) was constructed using this method and the resulting several mutants lacked both production of conidia and aflatoxin biosynthesis capacity. Southern blotting analysis demonstrated that the majority of the transformants contained a single T-DNA insert on the genome. To the best of our knowledge, this is the first report of genetic transformation of A. flavus via ATMT and our protocol provides an effective tool for construction of genome-wide gene mutant libraries for functional analysis of important genes in A. flavus.     

Genetic transformation and full recovery of alfalfa plants via secondary somatic embryogenesis

A genetic transformation method via secondary somatic embryogenesis was developed for alfalfa (Medicago sativa L.). Mature somatic embryos of alfalfa were infected by Agrobacterium strain GV3101 containing the binary vector pCAMBIA2301. pCAMBIA2301 harbors the uidA Gus reporter gene and npt II acts as the selectable marker gene. Infected primary embryos were placed on SH2K medium containing plant growth regulators to induce cell dedifferentiation and embryogenesis under 75 mg/L kanamycin selection. The induced calli were transferred to plant medium free of plant growth regulators for embryo formation while maintaining selection. Somatic embryos germinated normally upon transfer to a germination medium. Plants were recovered and grown in a tissue culture room before transfer to a greenhouse. Histochemical analysis showed high levels of GUS activity in secondary somatic embryos and in different organs of plants recovered from secondary somatic embryos. The presence and stable integration of transgenes in recovered plants were confirmed by polymerase chain reaction using transgene-specific primers and Southern blot hybridization using the npt II gene probe. The average transformation efficiency achieved via secondary somatic embryogenesis was 15.2%. The selection for transformation throughout the cell dedifferentiation and embryogenic callus induction phases was very effective, and no regenerated plants escaped the selection procedure. Alfalfa transformation is usually achieved through somatic embryogenesis using different organs of developed plants. Use of somatic embryos as explants for transformation can avoid the plant development phase, providing a faster procedure for introduction of new traits and facilitates further engineering of previously transformed lines.     

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation.In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.     

Aspergillus terreus Accessory Conidia Are Unique in Surface Architecture,Cell Wall Composition and Germination Kinetics

Infection with Aspergillus terreus is more likely to result in invasive, disseminated disease when compared to other Aspergillus species; importantly this species appears to be less susceptible to the antifungal drug amphotericin B. Unique to this species is the ability to produce specialized structures denoted as accessory conidia (AC) directly on hyphae both in vitro and in vivo. With the hypothesis that production of AC by A. terreus may enhance virulence of this organism, we analyzed the phenotype, structure and metabolic potential of these conidia. Comparison of A. terreus phialidic conidia (conidia that arise from conidiophores, PC) and AC architecture by electron microscopy revealed distinct morphological differences between the two conidial forms; AC have a smoother, thicker outer cell surface with no apparent pigment-like layer. Further, AC germinated rapidly, had enhanced adherence to microspheres, and were metabolically more active compared to PC. Additionally, AC contained less cell membrane ergosterol, which correlated with decreased susceptibility to AMB as determined using a flow cytometry based analysis. Furthermore, AC exhibited surface patches of β1-3 glucan, suggestive of attachment scarring. Collectively, the findings of this study suggest a possible role for AC in A. terreus pathogenesis.     

A novel chloroplast transformation vector compatible with the Gateway® recombination cloning technology

To analyze the suitability of Gateway^® vectors for transformation of chloroplasts, we converted a standard plastid transformation vector into a Gateway^® destination vector containing the necessary recombination sites attR1 and attR2. Insertion of the green fluorescent protein (GFP) coding sequence with associated T7g10 ribosome binding site into this destination vector created the expression vector for transformation of tobacco chloroplasts with the biolistic method. Correct integration of the transgene into the plastid genome was verified by PCR and the homoplasmic nature of the transformed plants was confirmed by Southern Blot analysis. Expression of the GFP reporter protein was monitored by confocal laser scanning microscopy (CLSM) and quantification by western blot analysis showed a GFP accumulation level of 3 % total soluble protein (TSP). The presented results clearly demonstrate that the Gateway^® recombination sites are compatible with all steps of plastid transformation, from generation of transplastomic plants to expression of GFP. This is the first report of a plastid transformation vector made by the Gateway^® recombinant cloning technology, which proves the suitability of this system for use in chloroplasts.