Bipolaris euphorbiae as a Biological Control Agent for Wild Poinsettia (Euphorbia heterophylla): Host-Specificity and Variability in Pathogen and Host Populations

Bipolaris euphorbiae (anamorphic Ascomycota: Pleosporaceae) has been studied since the early 1980s as a potential biocontrol agent of wild poinsettia (Euphorbia heterophylla), a noxious invader of soybean fields in Brazil. A new isolate (KLN05) was arbitrarily selected after the aggressiveness of six isolates of the fungus obtained from different sites was evaluated on wild poinsettias grown from seeds obtained from six different geographic origins in Brazil: Viçosa-state of Minas Gerais; Itabuna-state of Bahia; Niterói-state of Rio de Janeiro; Nova Laranjeira, Londrina and Foz do Iguaçú-state of Paraná. Bipolaris euphorbiae grew between 10 and 30 °C, with the optimum for colony growth at 25 °C. The best temperature range for conidial germination was between 20 and 30 °C. Conidial germination was not inhibited by mixing conidia with imazethapyr (106 g of active ingredient [a.i.]/l), glyphosate (720 g of a.i./kg), fomesafen (250 g of a.i./l), carfentrazone (400 g of a.i./l), atrazine (500 g of a.i./l) and glyphosate+carfentrazone (720 g of a.i/kg+400 g of a.i/l). Bipolaris euphorbiae was inoculated on plants belonging to 32 species of 12 different plant families and was only capable of infecting E. heterophylla.     

Comparative total and proportional rate of germination of Bipolaris sorokiniana and Curvularia geniculata conidia is influenced by culture age and temperature

Comparative observations of Bipolaris sorokiniana and Curvularia geniculata conidia germination as influenced by culture age and temperature showed some distinct differences, but generally established the ability of these organisms to function under similar conditions. Total germination of B. sorokiniana conidia was favored by increasing culture age from 20 to 60 days and temperature to 25°C; total conidia germination of C. geniculata was favored by increasing temperature to 25°C, but increasing culture age decreased germination. These reactions seem associated with conidia age. Maximum proportional intra-population germination of conidia of each organism also varied with culture age and temperature. At temperatures of 5°C and 15°C, amplitude of maximum proportional germination of both organisms increased as culture age was increased from 20 to 40 days and then decreased among 60-day-old cultures. At 25°C and above, amplitude of maximum proportional germination of conidia of both organisms decreased from each older culture. Progressively increasing temperature at a given culture age increased the amplitude of maximum proportional germination up to 25°C for conidia of B. sorokiniana, but generally decreased it for conidia of C. geniculata (except 20-day-old cultures). Frequency (specific 2 h interval) of maximum proportional intrapopulation germination of B. sorokiniana shifted from 6 h to 2 h in response to increasing temperature and culture age; conidia from youngest cultures of C. geniculata shifted to intervals of 4 h and 2 h in response to increasing temperature to 25°C, but among conidia from 60-day-old cultures, frequency shifted to 6 h intervals at all temperatures. Above 25°C, maximum proportional germination of C. geniculata conidia from cultures of all ages occurred at 6 h. It was concluded that the germination response of B. sorokiniana and C. geniculata conidia to temperature and culture age (and, subsequently, conidia age) are enough similar that these organisms could function in a potential ‘disease complex’ on Poa pratensis and Agrostis palustris.     

Effect of temperature on germination,growth, and infectivity of the mosquito pathogen Tolypocladium cylindrosporum (deuteromycotina; hyphomycetes)

The mosquito pathogen Tolypocladium cylindrosporum was examined with regard to its response to temperature. Similar temperature ranges were found for growth, germination, and infectivity of blastospores and conidia. Germination occurred at 8° and 33°C but not at 6° and 35°C. Optimal germination and growth was noted between 24° and 27°C for both spore types. Infectivity of blastospores and conidia at different temperatures was examined by exposing L₂Aedes sierrensis larvae to concentrations of 5 × 10⁵ blastospores/ml or 5 × 10⁶ conidia/ml. Larvae were incubated at 12°, 15°, 25°, and 30°C. Infection occurred at all temperatures tested with LT₅₀ values ranging from 22.7 days (12°C) to 5.6 (25°C) days for conidia and 4.7 days (12°C) to 0.6 day (25°C) for blastospores. These results confirmed earlier findings that blastospores infected and killed host larvae more rapidly than conidia and suggested that this difference is largely due to the more rapid germination rate of blastospores. These experiments demonstrated that T. cylindrosporum can be active against mosquito larvae over a broad range of temperatures encompassing both the cold-water habitat of certain temperate mosquito species as well as the habitat of tropical vector species.     

Selection of indigenous isolates of entomopathogenic soil fungus Metarhizium anisopliae under laboratory conditions

Eight native isolates of the entomopathogenic fungus Metarhizium anisopliae (Metschnikoff) Sorokin were obtained by monitoring soils cultivated in a conventional manner. These isolates were compared in three areas: (a) conidial germination, (b) radial growth and sporulation and (c) ability of conidia to infect Tenebrio molitor larvae. All bioassays were carried out at constant temperatures of 10, 15, and 20 °C. Conidia of individual isolates demonstrated differences in germination after a 24-h long incubation at all evaluated temperatures. At 20 °C, the germination ranged from 67 to 100 % and at 15 °C from 5.33 to 46.67 %. At 10 °C, no germination was observed after 24 h; nevertheless, it was 8.67–44.67 % after 48 h. In terms of radial growth, the culture diameters and the associated production of spores of all isolates increased with increasing temperature. At 10 °C, sporulation was observed in three isolates while all remaining cultures appeared sterile. Three weeks post-inoculation, conidia of all assessed isolates caused 100 % cumulative mortality of treated larvae of T. molitor at 15 and 20 °C with the exception of isolate 110108 that induced 81.33 % mortality at 15 °C. At 10 °C, larval cumulative mortality ranged from 6.67 to 85.33 % depending on the isolate. Isolates 110108 and 110111 showed significantly slower outset and a much lower rate of infection at all temperatures compared to other tested isolates of M. anisopliae. The bioassays were carried out with the purpose to sort and select indigenous isolates of M. anisopliae useful as biocontrol agents in their original habitat.     

Effect of Light, Temperature and Substrate during Spore Formation on the Germinability of Conidia of Colletotrichum falcatum

This paper presents results on the effect of light, temperature and substrate during spore formation on the germinability of conidia in Colletotrichum falcatum. Light seems to have no effect on the germination of conidia unless the cultures were exposed to a high intensity of light during sporulation, in which case the spores showed a reduced germination and an increased appressoria formation. Conidia produced at temperatures higher than the optimum showed better germination and less appressoria formation than the spores produced at the temperature optimum for the growth and sporulation of the fungus. A similar increase in germination was also observed in conidia obtained from inoculated sugarcane leaves as compared to those produced on culture media. The light type virulent isolates of C. falcatum showed greater sensitivity to all these treatments than the dark type weakly pathogenic isolates.     

Aggressiveness analysis of foliar fungi to leaves of wheat and rice crops

Abstract

The investigations were based on two surveys of wheat and one survey of rice. Alternaria alternata, Curvularia lunata, Helminthosporium tetramera and Bipolaris sorokiniana were isolated and identified from foliage and soil of both wheat and rice crops and their aggressiveness was studied by aggressiveness analysis screened out into different aggressiveness classes. The aggressiveness of isolated fungi was studied on wheat varieties (Inqalab-91 and Chakwal-86) and rice varieties (Basmati-385 and IRRI-6) under controlled conditions. In the foliar aggressiveness test of A. alternata, the overall number of aggressive isolates was higher on wheat varieties than rice. Bipolaris sorokiniana isolates showed foliar blight symptoms on wheat but not on rice varieties. In C. lunata, the overall number of aggressive isolates was higher on wheat. In the foliar aggressiveness test of H. tetramera, the number of non-aggressive isolates was almost the same on wheat and rice varieties. In the present study it became clear that A. alternate, B. sorokiniana, C. lunata and H. tetramera are common foliar pathogens in rice and wheat crops and can cause soil-borne and foliar diseases.     

Sensitivity of the Entomogenous Fungus Beauveria bassiana to Selected Plant Growth Regulators and Spray Additives

Mefluidide was the only one of four plant growth regulators that caused little to no significant inhibition of in vitro germination and growth of the entomogenous fungus Beauveria bassiana. Silaid, paclobutrazol, and flurprimidol significantly inhibited germination and growth. Mortality of fall armyworm, Spodoptera frugiperda, resulting from B. bassiana was significantly reduced when larvae were exposed to conidia plus soil treated with paclobutrazol. Larval mortality resulting from conidia plus soil treated with mefluidide did not differ significantly from mortality resulting from untreated conidia. Triton CS-7 was the only one of eight spray adjuvants that significantly inhibited germination of B. bassiana conidia.     

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.     

Laboratory assessment of entomopathogenic nematode symbiotic bacteria to control maize pest,Ostrinia furnacalis,and fungi diseases,Bipolaris maydis and Curvularia lunata

The application of entomopathogenic nematodes (EPN) and their symbiotic bacteria as biological control approaches depend on their lethal parasites to pest and antifungal activities against plant pathogenic fungi. We have collected 23 symbiotic bacterial strains from 23 EPN isolates gathered from different regions of China. In the present study, the insecticidal and antifungal activities of all these bacterial isolates were evaluated in the laboratory. Bioassay results showed that the broth and crude extract of all these 23 EPN symbiotic bacteria strains have, to a certain extent, oral insecticidal activity and/or growth inhibition to the larvae of Ostrinia furnacalis and antifungal activity against Bipolaris maydis and Curvularia lunata. Among these strains, SY5 exhibited highest insecticidal and antifungal activities to O. furnacalis, B. maydis and C. lunata. The adversity resistance of strain SY5 showed that the antifungal activity of the broth was more stable than the insecticidal activity, and the stability of antifungal activity to B. maydis and C. lunata was different.     

Optimising inoculum yield and shelf life of Plectosphaerella cucumerina,a potential bioherbicide for Cirsium arvense

Plectosphaerella cucumerina was identified as a potential bioherbicide for controlling Cirsium arvense in Canada and New Zealand. The current study evaluated production conditions using two isolates (one from each country) to determine whether the yield and shelf life of inoculum are suitable for mass production. Mycelial growth and sporulation in culture both increased from 15°C to 25°C and declined at higher temperatures with no mycelial growth at 37°C. The Canadian isolate produced fewer conidia than a New Zealand isolate. Potato dextrose-based liquid media with moderate to high concentrations of carbohydrates (25%, 50%, and 100%) maximised conidia production and these base media produced conidia with the highest germination rate (>80%) both at harvest and after 4 weeks stored at 4°C in 2.5% glycerol, 40% milk glycerol or after air drying. However, after 10-week storage, the conidia failed to germinate. Sporulation occurred during growth on all solid substrates tested (rice, rolled barley, and triticale), but conidial germination was highest on rice and barley, both before and after air drying. By contrast to conidia, 90% of mycelia-infested barley grains were viable after 3 years of storage at room temperature, although viability was lost by this time on the other substrates. This study has shown that the nutritional base is an important determinant of sporulation and shelf life for P. cucumerina. Although the yield of conidia in liquid medium was adequate to justify further development of P. cucumerina as a bioherbicide, improvement in its shelf life, or alternate formulation types that extend the shelf life, must be made for commercial efficiency.     

The effect of the formulation on the shelf-life of biopesticides based on two colombian isolates of Trichoderma koningiopsis Th003 and Trichoderma asperellum Th034

BackgroundFour biopesticide prototypes formulated as dispersible granules and dry powders based on 2 Colombian isolates of Trichoderma koningiopsis (Th003) and T. asperellum (Th034) were developed. These microorganisms have antagonist activity against Fusarium oxysporum f. sp. lycopersici and Rhizoctonia solani with a reduction in incidence of between 70 and 100% in tomato crops and potato crops, respectively.AimTo determine the effect of the formulation on the shelf-life of 4 biopesticides based on T. koningiopsis Th003 and Trichoderma asperellum Th034 at 3 different temperatures.MethodsThe formulation effect was determined by evaluating the germination of unformulated and formulated conidia (dispersible granules and dry powder) stored at 8, 18 and 28 °C for 18 months. Germination kinetics were used to estimate the shelf-life by using different mathematical models (zero order, first order, second order, Higuchi model, Korsmeyer-Peppas model and polynomial model).ResultsThe products showed high stability of the conidia germination when they were stored at 8 and 18° C, with shelf-lives of 14.4 and 13.9 months for dry powder based on Th003, and 12.0 and 10.8 months for dry powder based on Th034, respectively. Prototypes formulated as dispersible granules stored at the same temperatures (8 and 18 °C) showed lower shelf-lives, with values of 11.6 and 10.9 months for the Th003 product, and 10.7 and 7.2 months for the dispersible granules based on Th034. Significant reductions in germination were observed on unformulated conidia at all storage temperatures evaluated.ConclusionsThe formulation type affected the conidia stability of the 2 Trichoderma spp. Colombian isolates. Dry powder was the prototype with the highest stability and shelf-life at all temperatures evaluated.     

Selective isolation of dematiaceous fungi from the workers of <Emphasis Type="Italic">Atta laevigata</Emphasis> (Formicidae: Attini)

Leaf-cutting ants (Formicidae: Attini) are considered pests in agriculture for their impact in human crops, as they utilize leaf fragments to raise their fungal mutualist (Agaricales: Lepiotaceae). Basically, the basidiomycetous fungus is cultivated to supply food to adult workers and broads; in return, the ants protect it against natural enemies. However, recent studies have claimed that other microorganisms are associated to ant nests where a wide range of interactions may take place. To investigate the occurrence of dematiaceous fungi on the cuticle of Atta laevigata ants, 30 workers were sampled from an adult nest located in the surroundings of the Center for the Studies of Social Insects, UNESP-Rio Claro, SP, Brazil. The use of selective techniques to avoid high-sporulation fungi has been recommended and was tested in this study. To favor the isolation of the desired fungi, heads and cuticle scrapings of ant bodies were inoculated on Mycosel agar and incubated for 3 weeks at 35°C. Morphological and molecular methods were used to identify the filamentous fungi recovered. From 56 isolates, 19 were hyaline filamentous species, and among the remaining 37, some are mentioned as phyto-associated fungi like Alternaria arborescens, Bipolaris sorokiniana, Bipolaris eleusines, Bipolaris zeae, Curvularia trifolii, and Paraphaeosphaeria michotii. These species are reported from A. laevigata bodies for the first time. None of the isolation trials revealed the presence of the parasite Escovopsis or entomopathogenic fungi. The possible spread of the fungi in nature by the ants is discussed.     

Effect of constant temperatures on germination, radial growth and virulence of Metarhizium anisopliae to three species of African tephritid fruit flies

The effect of temperatureon conidial germination, mycelial growth, andsusceptibility of adults of three tephritidfruit flies, Ceratitis capitata(Wiedemann), C. fasciventris (Bezzi) andC. cosyra (Walker) to six isolatesof Metarhizium anisopliae were studied inthe laboratory. There were significantdifferences among the isolates in the effect oftemperature on both germination and growth.Over 80% of conidia germinated at 20, 25 and30°C, while between 26 and 67% conidiagerminated at 35°C and less than 10% at15°C within 24 hours. Radial growth was slowat 15°C and 35°C with all of theisolates. The optimum temperature forgermination and mycelial growth was 25°C. Mortality caused by the six fungal isolatesagainst the three fruit fly species varied withtemperature, isolate, and fruit fly species.Fungal isolates were more effective at 25, 30and 35°C than at 20°C. The LT₉₀values decreased with increasing temperature upto the optimum temperature of 30°C. Therewere significant differences in susceptibilitybetween fly species to fungal infection at allthe temperatures tested.     

Effects of nutrient medium composition and temperature on the germination of conidia and the entomopathogenic activity of the fungi Beauveria bassiana and Metarhizium anisopliae

The effects of nutrient medium composition and temperature on the germination of conidia of the fungi Beauveria bassiana (strain AlG) and Metarhizium anisopliae (strain M-99) and their entomopathogenic activity have been studied. It was demonstrated that the presence of carbohydrates alone was sufficient for the spores of M. anisopliae M-99 to germinate, whereas the germination of B. bassiana AlG spores was inhibited by carbohydrates. Addition of KJ, ZnSO₄, or KBr into the Czapek medium increased the entomopathogenic activity of B. bassiana. The optimum temperature for spore germination was 20–35°C in both fungal species.     

Natural variation of ascospore and conidial germination by Fusarium verticillioides and other Fusarium species

Fusarium verticillioides and other Fusarium species were examined for their spore germination phenotypes. In general, germinating spores of F. verticillioides formed germ tubes that immediately penetrated into agar. Such invasive germination was the predominant growth phenotype among 22 examined field isolates of F. verticillioides from a broad range hosts and locations. However, two of the field isolates were unique in that they formed conidial germ tubes and hyphae that grew along the surface of agar before penetration eventually occurred. Conidia of 22 other Fusarium species were assessed for their germination phenotypes, and only some strains of F. annulatum, F. fujikuroi, F. globosum, F. nygamai, and F. pseudoanthophilum had the surface germination phenotype (21 % of the strains assessed). Sexual crosses and segregation analyses involving one of the F. verticillioides surface germination strains, NRRL 25059, indicated a single locus, designated SIG1 (surface vs. invasive germination), controlled the germ tube growth phenotypes exhibited by both conidia and ascospores. Perfect correlation was observed between an ascospore germination phenotype and the germination phenotype of the conidia produced from the resulting ascospore-derived colony. Recombination data suggested SIG1 was linked (7 % recombination frequency) to FPH1, a recently described locus necessary for enteroblastic conidiogenesis. Corn seedling blight assays indicated surface germinating strains of F. verticillioides were less virulent than invasively germinating strains. Assays also indicated pathogenicity segregated independently of the FPH1 locus. Invasive germination is proposed as the dominant form of spore germination among Fusarium species. Furthermore, conidia were not necessary for corn seedling disease development, but invasive germination may have enhanced the virulence of conidiating strains.     

Production, Partial Purification and Characterization of Extracelluar Xylanase from Chaetomium globosum

Culture conditions for efficient production of extracellular xylanase by fungus, Chaetomium globosum isolate Cg2, have been standardized. Further, xylanase has been partially purified and characterized. Xylanase activity was maximum after 9 days of incubation when amended in medium with 1.5 % xylan as carbon source and 0.6% NH₄H₂PO₄ as nitrogen source. Partial purification of the xylanase was accomplished by ammonium sulphate precipitation, followed by further purification by anion exchange chromatography on DEAE-Sephadex A-50 column. The partially purified enzyme was electrophoresed on SDS-PAGE and a single band produced corresponded to molecular weight, 32 kD. The optimum temperature and pH for maximum activity of purified xylanase were 30°C and 5.5, respectively. Both the purified xylanase and culture filtrate have shown the antifungal activity against Bipolaris sorokiniana, a causal organism of spot blotch of wheat. Purified xylanase at 100 μg ml^?1 concentration caused 100 per cent inhibition of conidia germination of B. sorokiniana, whereas the culture filtrate was able to inhibit germination up to 67.5 per cent.     

Thermal response and horizontal transmission of cameroonian isolates of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae – Candidates for microbial controls of the banana root borer Cosmopolites sordidus

Beauveria bassiana and Metarhizium anisopliae are two promising microbial agents for biopesticides development against the banana root borer Cosmopolites sordidus. In this study, germination, mycelial growth, and sporulation of six local Cameroonian isolates of those two species were assessed under seven different thermal conditions (13, 15, 20, 25, 29 33, and 37 °C) to select thermo-tolerant isolates. The Transmission potential of the thermo-tolerant isolates was determined at 25 ± 1 °C by dipping adult weevils in conidial suspensions (3.2 × 10⁸) conidia/ml and mixing these with uninoculated weevils in different proportions (0, 10, 30 and 50%), in groups of 30, and assessing the spread of the mycosis within the group over 35 d of co-incubation. Incubation temperature and isolates significantly affected germination, mycelial growth and conidial production. All isolates had large thermal tolerance ranges (13–33 °C) except MIITAC6.4.2 (20–29 °C). Horizontal transmission resulted in mortality of non-inoculated weevils from 4.63 ± 1.77 to 53.3 ± 11.9%. The isolate BIITAC6.2.2 exhibited high auto-dissemination potential and high conidia yield in cadavers. These results demonstrate the potential use of these isolates for biopesticides development against C. sordidus in Central Africa.     

Temperature effects on hyphal growth of wood-decay basidiomycetes isolated from Pinus densiflora deadwood

Hyphal growth rates were tested on malt extract agar plates at eight different temperatures (5–40?°C) using 36 isolates of 17 basidiomycete species obtained from Pinus densiflora deadwood in Japan. All isolates of four brown rot species showed optimum growth at 30?°C, whereas the optimum growth temperature of white rot species varied from 20?°C to 30?°C. Analysis using a dataset from four cooler sites showed that brown rot fungi grew more rapidly than white rot fungi at higher temperatures (25?°C, 30?°C, and 35?°C). These results suggest that the hyphal growth of brown rot fungi might be physiologically adapted to higher temperatures than those of white rot fungi among the fungal species inhabiting deadwood of P. densiflora in Japan.     

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.     

Methylobacterium Species Promoting Rice and Barley Growth and Interaction Specificity Revealed with Whole-Cell Matrix-Assisted Laser Desorption / Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF/MS) Analysis

Methylobacterium species frequently inhabit plant surfaces and are able to utilize the methanol emitted from plants as carbon and energy sources. As some of the Methylobacterium species are known to promote plant growth, significant attention has been paid to the mechanism of growth promotion and the specificity of plant–microbe interactions. By screening our Methylobacterium isolate collection for the high growth promotion effect in vitro, we selected some candidates for field and pot growth tests for rice and barley, respectively. We found that inoculation resulted in better ripening of rice seeds, and increased the size of barley grains but not the total yield. In addition, using whole-cell matrix-assister laser desorption/ionization- time-of-flight mass spectrometry (MALDI-TOF/MS) analysis, we identified and classified Methylobacterium isolates from Methylobacterium-inoculated rice plants. The inoculated species could not be recovered from the rice plants, and in some cases, the Methylobacterium community structure was affected by the inoculation, but not with predomination of the inoculated species. The isolates from non-inoculated barley of various cultivars grown in the same field fell into just two species. These results suggest that there is a strong selection pressure at the species level of Methylobacterium residing on a given plant species, and that selection of appropriate species that can persist on the plant is important to achieve growth promotion.