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.     

The effects of culture media, solid substrates, and relative humidity on growth, sporulation and conidial discharge of Valdensinia heterodoxa

Valdensinia heterodoxa (Sclerotiniacae) is a potential fungal bioherbicide for control of salal (Gaultheria shallon). The effect of culture media, substrates and relative humidity (RH) on growth, sporulation and conidial discharge of V. heterodoxa was determined for two isolates PFC2761 and PFC3027 in vitro. Culture media significantly affected the growth, sporulation, and conidial discharge of V. heterodoxa. Of eight agar media used, colony radial growth was optimal on salal oatmeal agar and salal potato dextrose agar for isolates PFC2761 and PFC3027, respectively; whereas sporulation was at an optimum on salal oatmeal agar for both isolates. Of the eight liquid media tested, mycelial production was highest on wheat bran–salal–potato dextrose broth. Growth on solid substrates greatly stimulated sporulation and conidial discharge of V. heterodoxa. Of the 12 solid substrates used, the greatest numbers of discharged conidia were observed from wheat bran and wheat bran–salal within 14 d of sporulation. Sporulation on solid substrates continued for 42 d. RH significantly affected the sporulation and conidial discharge for both isolates across all solid substrates tested. No conidia were produced or discharged below 93 % RH on wheat bran–salal and millet. With an increase of the RH from 93 to 97 %, sporulation and the number of discharged conidia increased significantly for both isolates on wheat bran–salal, but not on millet.     

Effect of temperature and water activity on mycelia radial growth,conidial production and germination of Lecanicillium spp. isolates and their virulence against Trialeurodes vaporariorum on tomato plants

Biocontrol of the whitefly Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae) using entomopathogenic fungi has been a difficult challenge under greenhouse conditions. In order to select fungal isolates adapted to high temperature and extremely low moisture nine isolates of Lecanicillium lecanii (Zimmerman) Zare & W. Gams, L. attenuatum Zare & W. Gams and L. longisporum (Petch) Zare & W. Gams (Hypocreales: Clavicipitaceae) were evaluated. In vitro assays were performed to determine colony radial growth, conidial production and conidial germination in three water activity media (a_w = 0.97, 0.98 and 1.00) at 28 and 32 °C. Virulence of Lecanicillium spp. isolates was evaluated against third instar T. vaporariorum on tomato plants at 23 °C. Colony radial growth, conidial production and germination decreased with the reduction in water activity, while 32 °C was extremely detrimental for all fungal isolates. However, some isolates were able to grow and produce conidia at low water activity and high temperature. Additionally, mortality above 60 % was recorded for one of these isolates. Practical implementation of biocontrol of T. vaporariorum under greenhouse production systems should consider the selection of those Lecanicillium isolates that show tolerance to the adverse environmental conditions in greenhouses.     

Nuclear distribution in Alternaria tenuis

Nuclear distribution and behaviour during vegetative growth and spore formation inAlternaria tenuis was studied utilising the HCl — Giemsa staining technique. The vegetative mycelium and conidia are predominantly monokaryotic. Anastomoses, followed by nuclear migrations, have been recorded. Intercellular nuclear migrations have only been observed in germinating conidia. Nuclear behaviour during conidial formation indicates that the conidia are homokaryotic. Cytological differences have been found in different monocoidal isolates of A. tenuis.     

Effect of relative humidity,temperature and fungicide on germination of conidia of Cercospora canescens caused the Cercospora leaf spot disease in mungbean

The aim of the present investigation was to determine the impact of relative humidity (RH) and temperature on conidial germination, nuclear position and effect of important fungicides on growth and conidial germination of Cercospora canescens. Germination of conidia was observed at RH range 92–100% at 5–35°C. Significant interaction between temperature and RH indicated that higher humidity and high temperature promoted quick germination both in the presence and absence of free moisture. Although in absence of free moisture at 92–95% RH higher temperatures 25–35°C promoted quick evaporation of moisture and no conidial germination. Number of germtube was increased significantly at the optimum temperature 25–30°C and higher humidity (98–100%). But higher temperature 25–35°C with lower RH did not support the conidial germination. This finding is very important for disease forecasting using meteorological data. The spray of Carbendazim as contact fungicide may not be useful since it is not effective against the conidia of C. canescens. Triadimefon did not inhibit the conidia germination but completely inhibited mycelium development at 50 μg/ml. Propriconazole inhibited both conidia germination and mycelial development. Therefore, Propiconazole may be taken as protective as well as curative spray. In non-systemic fungicide, Copper oxychloride gave anticipated result by inhibiting both conidial germination and mycelium development. Therefore, copper oxychloride can be used as protectant fungicides for Cercospora leaf spot caused by C. canescens.     

Effect of a membrane interactive peptide on plant cells of canola (Brassica napus) and two fungal pathogens

Summary A membrane interactive peptide was toxic to microspores, pollen and protoplasts of canola in the 1–5 µM concentration range. Similarly, at 5.0 µM the peptide completely inhibited germination of conidia ofVerticillium albo-atrum; however, when tested with conidia of a virulent isolate of blackleg (Leptosphaeria maculens), a fungal pathogen of canola, much higher levels (>30 µM) of the peptide were required to reduce or arrest germination and growth of the conidia. When testing the relative toxicities of novel peptides on plant cells and their pathogens, pollen germination is a simple, rapid and reliable alternative to protoplasts.     

Susceptibility of the cattle tick Boophilus microplus (Acari: Ixodidae) to isolates of the fungus Beauveria bassiana

The susceptibility of the tick Boophilus microplus to Beauveria bassiana was evaluated by inoculating eggs, larvae and engorged females of the tick with five fungal isolates at concentrations of 10⁶, 10⁷ and 10⁸ conidia/ml. Tick eggs (0.25 g) were immersed in 1 ml of a suspension of the different conidial concentrations for 1 min. Similar exposure was performed by immersion of 2000 larvae and homogeneous groups of nine engorged females in 2 and 20 ml of conidial suspension, respectively. Treated eggs, larvae and adults were placed in an incubator at 27 ± 1 °C and relative humidity above 80% for evaluation of the fungal action. All fungal isolates applied at all conidial concentrations reduced the hatching rate of larvae from treated eggs by 1.36–65.58% and increased the mortality rate of inoculated larvae by 0.8–70.49%. In the bioassay with engorged females, oviposition period was reduced by 9.69–47.80%, egg mass weight by 4.71–53.87%, estimated reproduction by 8.3–60.62%, egg production index by 5.03–54.20%, percent larval hatching by 0.27–13.96%, and the mortality rate of treated females was increased by 96.60–100%. The reduction of the estimated reproduction obtained for the treated groups ranged from 8.37 to 64.52%. The sporulation of the pathogen on dead females ranged from 3.70 to 88.88% depending on the isolate and concentration used. Isolates AM 09, CB 7 and JAB 07 were the most effective and effectiveness increased with increasing concentrations of conidia in the suspensions.     

Successful induction and recognition of conidiation,conidial germination and chlamydospore formation in pure culture of Morchella

Morels, fungi from the genus Morchella, are popular edible mushrooms. However, little knowledge of their asexual reproduction and inaccessible pure mitospores hamper illumination of their life cycle. Herein, we successfully induced conidiation, conidial germination and chlamydospore formation in pure culture of Morchella sextelata. Conidiation proceeded via four morphologically distinct stages: development of the conidiophore stalk, stalk branching, phialide differentiation, and conidium production. Terminal and intercalary chlamydospores were formed on conidial hyphae. The development of conidiophores occurred earlier, with more conidia produced, in cross-mating cultures than in single-spore cultures. Mature conidia were spherical and 2.5–8 μm in diameter, with a vast majority (nearly 99%) 2.5–5 μm in diameter. Each conidium contained one to three nuclei (80.2% conidia contained one nucleus, 19.1% contained two nuclei, and 0.7% contained three nuclei). The conidial nucleus diameter was 1–2 μm. The nuclear mitosis in detached conidia that was observed may benefit their colony initiation. Additionally, morel conidia formed conidial anastomosis tubes. Conidia (mitospores) likely not only function as spermatia, but also as reproductive propagules in Morchella. Further research is imperative to elucidate the relationship between the conidia and chlamydospores, and their unique function in the morel life cycle.     

Phylloplane microorganisms as a potential biocontrol agent against Helminthosporium oryzae Breda de Hann,the incitant of rice brown spot

Abstract

Fifteen pathogenic isolates of Helminthosporium oryzae were established from 15 different rice growing areas representing nine districts: Madurai, Theni, Thanjavur, Thiruvarur, Nagapatinum, Tirunelveli, Trichy, Ariyalur and Cuddalore district of Tamil Nadu. The symptoms initially appeared on the leaves as small, oval, dark brown to black spots. Under favourable conditions, the fungus attacks grain and caused grain discoloration. The isolate, collected from Ammapettai of Thanjavur district was the most virulent (Grade 7.47, PDI 82.96) on rice plants followed by Trichy isolate I₂ (Grade 7.26; PDI 80.74) while I₁₅ (Cumbum) was the least virulent (Grade 1.8, PDI 20.00). The isolates of H. oryzae varied in size (length and width) of the conidia, colour and the number of cells per conidium. The maximum (39.1 μm) length of the conidium was observed in I₁₀ followed by I₁₁ (37.3 μm). The maximum (17.1 μm) conidial width was observed in I₈. The colour of the conidium varied from light brown (isolates I₃, I₆ and I₁₄) to brown, while there was no appreciable difference in the conidial shape. The number of cells varied from 2 – 4 among the isolates. Six phylloplane microorganisms were tested against the virulent isolate of H. oryzae. Among them, Cladasporium spp was very effective in inhibiting the mycelial growth and spore germination of H. oryzae followed by Penicillum spp and Aspergillus flavus while Bacillus subtilis was least effective in inhibiting the mycelial growth and spore germination of H. oryzae.     

Factors influencing pathogenicity of Fusarium tumidum on gorse (Ulex europaeus)

Factors promoting pathogenicity of Fusarium tumidum on gorse (Ulex europaeus) were determined to develop a novel strategy for delivering this potential mycoherbicide using insects as vectors of inoculum. Fusarium tumidum sprayed as a suspension of 1×10⁶ conidia mL^?1 on at least 50% of a gorse plant reduced shoot dry weight by 45% (P<0.05). A minimum of 910 viable conidia were required to cause a lesion on leaves. The leaves and flowers were more susceptible to infection than stems, spines and pods. Generally, wounding of gorse leaves and stem increased F. tumidum infection, most likely through releasing nutrients that enhanced conidial germination and hyphal growth. We showed in a separate experiment that conidial germination (93%) and germ tube length (407 µm) were greater when incubated in 0.2% gorse extract solution for 24 h than in water (62% germination, germ tube length 42 µm). Inoculation of gorse with a F. tumidum conidial suspension supplemented with 0.2% gorse extract resulted in a shoot dry weight reduction (P=0.012) equivalent to that of plants that were wounded and inoculated. It is concluded that wounding of older tissues (which mimics insect damage) is required to facilitate F. tumidum infection of mature gorse plants.     

In Vitro and <Emphasis Type="Italic">In Planta</Emphasis> Compatibility of Insecticides and the Endophytic Entomopathogen, <Emphasis Type="Italic">Lecanicillium lecanii</Emphasis>

In an attempt to clarify the potential role of endophytic fungi in integrated pest management, the compatibility of an endophytic isolate of Lecanicillium lecanii (Zimmermann) Gams & Zare (Hyphomycetes) with nine insecticides used against Aphis gossypii Glover (Homoptera : Aphididae) was examined both in vitro over 14 days and in planta. In the laboratory, most insecticides partially or completely inhibited the germination of conidia and growth of hyphae in nutrient-rich conditions. Endosulfan completely inhibited the germination of conidia and hyphal growth. In contrast, all insecticides were compatible with L. lecanii in planta, and the fungus was readily recovered from inoculated, colonized leaves. These data support the hypothesis that endophytic L. lecanii will be unaffected by insecticides and could be integrated in the management of pests in cotton.     

Morphological characterization and optimization of conditions for conidial production of Elsinoë ampelina,the causal organism of grapevine anthracnose

Grape anthracnose, which is caused by Elsinoë ampelina, is a disease that negatively affects grape production. This study aimed to investigate the effects of aeration, temperature, light, and preculture period on the formation of E. ampelina conidia and conidial germination and virulence. The colony morphology on potato dextrose agar (PDA) plates was more diverse than that in PDA bottles. The assessment of different culture methods, temperatures, light conditions, and preculture periods revealed that optimal conidial production occurred on 25‐day‐old colonies grown in PDA bottles at 21°C for 24 hr in the dark. The cultures in PDA bottles consistently produced approximately 5.0 × 10⁶ conidia under these conditions. No conidial formation occurred when the cultures were kept at 25°C in the dark. The highest germination rate of E. ampelina was 80% at 25°C after 24 hr, whereas no germination was observed at 17°C after 12 hr. Pathogenicity tests revealed that symptoms of the disease were observed 4 days postinoculation (dpi) on leaves of Vitis vinifera cv. Red Globe. New conidia were observed on the lesions at 8 dpi. This study provides an effective method for the conidial production of E. ampelina that may also be applicable for other Elsinoë fungal species.     

Conidial germination and germ tube elongation of Phomopsis amaranthicola and Microsphaeropsis amaranthi on leaf surfaces of seven Amaranthus species: Implications for biological control

Microsphaeropsis amaranthi and Phomopsis amaranthicola are potential biological control agents for several Amaranthus species. In an effort to understand the initial infection processes with these pathogens, a study was conducted of the conidial germination and germ tube length (μm) on the weed leaf surfaces at 21 °C and 28 °C. Weeds included Amaranthus rudis, A. palmeri, A. powellii, A. retroflexus, A. spinosus, A. hybridus, and A. albus. For P. amaranthicola, conidial germination and germ tube length varied among the seven weed species at both temperatures, while for M. amaranthi the differences in germ tube lengths were significant among weed species only at 21 °C. While the conidia of M. amaranthi and P. amaranthicola germinated on the leaf surfaces of all seven weed species, temperature appeared to impact the number and length of germ tubes on the leaf surfaces. The percentage of germinated conidia and the length of germ tubes at both temperatures were often greater for M. amaranthi than for P. amaranthicola. In order for the fungal pathogen to successfully infect and kill a weedy host, conidia must germinate and form a germ tube, two processes that vary with host species and temperature for M. amaranthi and P. amaranthicola. The extent to which successive infection processes, e.g., penetration, invasion and colonization, contribute to host specificity warrants study.     

Conidial water affinity is an important characteristic for thermotolerance in entomopathogenic fungi

The interspecific thermotolerance of several species of entomopathogenic fungi was evaluated based on the conidial water affinity. The species were divided between hydrophilic and hydrophobic conidia. The species with hydrophobic conidia were Beauveria bassiana (ARSEF 252), Metarhizium brunneum (ARSEF 1187), Metarhizium robertsii (ARSEF 2575), Isaria fumosorosea (ARSEF 3889) and Metarhizium anisopliae s.l. (ARSEF 5749). The species with hydrophilic conidia were Tolypocladium cylindrosporum (ARSEF 3392), Tolypocladium inflatum (ARSEF 4877), Simplicillium lanosoniveum (ARSEF 6430), Lecanicillium aphanocladii (ARSEF 6433), S. lanosoniveum (ARSEF 6651), Aschersonia placenta (ARSEF 7637) and Aschersonia aleyrodis (ARSEF 10276). The conidial surface tension of each isolate was also studied. Conidial suspensions were exposed to 38, 41 or 45 °C. After exposure, the suspensions were inoculated on media and conidial germination was evaluated. Considerable differences in thermotolerance were found among the 12 entomopathogenic fungal species. Species with hydrophobic conidia were generally more thermotolerant than species with hydrophilic conidia. All isolates with hydrophobic conidia showed higher conidial surface tension than the isolates with hydrophilic conidia.     

Persistence and Viability of Lecanicillium lecanii in Chinese Agricultural Soil

The entomopathogenic fungus L. lecanii has been developed as biopesticides and used widely for biological control of several insects in agricultural practice. Due to the lack of isolation/count methods for L. lecanii in soil, the persistence of this fungus in soil appears to have attracted no attention. A selective medium and count method for L. lecanii in soil based on cetyl trimethyl ammonium bromide (CTAB) was developed, and then the persistence and viability of this fungus in soil were investigated under field conditions between 2012 and 2014. The results showed that the rate of recovery for L. lecanii in soil on the selective CTAB medium was satisfactory. The minimum CFUs for L. lecanii on the selective medium (0.5 g/L CTAB) was about 10² conidia/g soil. The L. lecanii density in soil declined quickly in the first month after inoculation with fungal conidia, kept stable for 6 to 10 months, and then decreased gradually until undetectable. L. lecanii could persist for at least 14 months in the agricultural soil of northern China. The colony growth, conidia yield and germination rate on plates, as well as the median lethal concentration or times (LC₅₀ or LT₅₀) to aphids, mycelium growth in aphids and sporulation on aphids of L. lecanii did not change significantly during the persistence in soil. In general, the count method developed here was a very useful tool for monitoring the dynamics of natural or introduced L. lecanii populations in soil, and the data on the persistence of L. lecanii in soil reported here were helpful for biological control and environmental risk assessment.     

Modelling the effects of temperature and wetness on the polycyclic phase of Stemphylium vesicarium,the pathogen causing purple spot on asparagus (Asparagus officinalis L.)

The polycyclic phase of Stemphylium vesicarium is the key factor for the forecast and integrated control of purple spot on asparagus. The annual dynamics of airborne conidia were determined under field conditions by conidia traps. From 2013 to 2015, conidia became airborne at the earliest at mid‐July, but the number trapped was considerably enhanced only after mid‐August, early September. The cumulative percentage of trapped conidia was best described using a logistic function depending on the daily temperature sum (base 0°C) accumulated only on days with >0.2 mm of rainfall (R² = .81). The germination of conidia was modelled by a generalized beta‐modified Chapman Richards function, and the germ tube length was modelled by a generalized beta‐power function. Conidia germinated in a wide temperature range, with an optimum at 23.3°C, whereas germ tube length had a narrow nearly optimum temperature range around 28.7°C, which indicates that infection by conidia is more restricted by germ tube growth than by germination. The effect of temperature on the number of lesions produced by two strains on green asparagus spears had the narrowest optimum range (optimum at 21.9°C) of all parts of the polycyclic phase. In plant tissue, the spread of the fungus depends on the mycelium growth. The mycelium growth of the four strains, which was modelled with data from a petri dish experiment, had an optimum temperature at 24.7°C.     

Relationship between number and type of adhesions of Penicillium oxalicum conidia to tomato roots and biological control of tomato wilt

We studied (a) the extent adhesion of Penicillium oxalicum conidia to tomato roots after application of P. oxalicum conidial formulations with or without stickers, (b) the relationship between the extent of conidial adhesion to roots and biocontrol of the conidial formulations against tomato wilt, and (c) colonisation of roots by P. oxalicum. Adhesion of P. oxalicum conidia to tomato roots occurred within the first minute of contact between the root and the conidial formulation and the bonding strength was sufficiently strong to prevent conidial removal from the roots. In addition, some formulations with stickers that increased conidial adhesion to roots improved the biocontrol of tomato wilt, when compared to that of formulations without stickers. A “dried conidia without stickers” with 0.025% Nu-Film 17 had no effect on the biocontrol of tomato wilt, despite good adherence of the conidia to the roots. The numbers of P. oxalicum conidia that adhered to the roots was constant for 60 days after application of a “dried conidia without stickers” conidial formulation. The significance of these results (speed of adhesion, number of adhered conidia, and variability of conidial external surface) are discussed in relation to the biocontrol success of tomato wilt using different types of conidial formulations with and without stickers.     

Aflatoxins in mutants of Aspergillus flavus

Mutants ofAspergillus flavus were recovered following the irradiation of conidia with ultraviolet light. Analysis of the mutants for aflatoxins B₁, B₂, G₁, and G₂ indicated a wide range of variability in aflatoxin levels. None of the isolates produced the G toxins, and four produced little or no aflatoxin B₂. Production of B₁ and B₂ by the mutants ranged from 1.3 µ;g/ml to 967 µg/ml and zero to 30 µg/ml, respectively. The correlation between production of B₁ and B₂ was statistically significant. There was no apparent correlation between nutritional requirement or conidial color and aflatoxin production.     

Impact of moisture on in vitro germination of Metarhizium anisopliae and Beauveria bassiana and their activity on Triatoma infestans

The in vitro germination of 11 Metarhizium anisopliae and 11 Beauveria bassiana isolates originating from substrates collected in rural peridomestic areas in Central Brazil where triatomines are common was tested. Conidia completed germination up to 24 h after exposure to water activity of >0.99 a_w in all isolates tested. At lower 0.93 a_w germination was delayed but conidia of most isolates germinated at high rates (>98 %) within 216 h of incubation. Activities of 2 M. anisopliae and 2 B. bassiana isolates with different patterns of germination at 0.93 a_w were tested in Triatoma infestans third instar nymphs. There was no relationship between germination kinetics in vitro at 0.93 a_w and their activity in vivo at 98, 75 and 43 % relative humidity (rh). Isolates with accelerated germination at 0.93 a_w were not more virulent at 75 and 43 % rh compared with isolates with retarded or no germination. Highest mortalities were observed at 98 % rh, and they did not exceed 25 % after 25 d incubation at lower 75 and 43 % rh. Isolates that originated from a region with an extensive annual arid period showed no adaptation to lower humidity in their activity against T. infestans.     

Susceptibility of the tobacco whitefly,Bemisia tabaci (Hemiptera: Aleyrodidae) biotype Q to entomopathogenic fungi

Recently, the Q biotype of tobacco whitefly has been recognized as the most hazardous strain of Bemisia tabaci worldwide because of increased resistance to some insecticide groups requiring alternative strategies for its control. We studied the susceptibility of this biotype of B. tabaci to 21 isolates of Beauveria bassiana, three isolates of Isaria fumosorosea, one isolate of I. cateni, three isolates of Lecanicillium lecanii, one isolate of L. attenuatum, and one isolate of Aschersonia aleyrodis. These isolates were evaluated on pruned eggplant seedlings, at a concentration of 10⁸ conidia/mL (deposited at 6000±586 conidia mm^?2). The mortality based on mycosis varied from 18 to 97% after 6 days. Isaria fumosorosea isolate Pf04, B. bassiana isolates Bb06, Bb12, and L. lecanii L14 were found the most effective. Furthermore, five isolates were chosen for concentration–mortality response assays and compared to B. bassiana GHA as a standard. The numbers of nymphs infected by fungi were correlated with the spore concentration. L. lecanii L14 and I. fumosorosea Pf04 had the shortest LT₅₀ at 3.5 and 3.3 days at 6000±586 conidia mm^?2. Mortality declined and LT₅₀ values were longer as the concentration of conidia was reduced. The LD₅₀ values were calculated as 87, 147, 191, 263, and 269 conidia mm^?2 for isolates L14, GHA, AS1260, Bb13, and Pf04, respectively. These results indicated that the Q biotype of sweetpotato whitefly was susceptible to the five isolates of entomopathogenic fungi and these isolates have potential to be developed as microbial pesticides for whitefly control.