Production and extraction of extracellular lipase from the entomopathogenic fungus Isaria fumosoroseus (Cordycipitaceae; Hypocreales)

Lipases are important cuticle degrading enzymes involved in the infection process of entomopathogens by hydrolysing the ester bonds of lipoproteins, fats and waxes present in the insect integument. Production of extracellular lipase by Isaria fumosoroseus (Cordycipitaceae; Hypocreales) isolate IF28.2 was investigated using different combinations of basal medium components. The effect of different vegetable oils added to a basal medium at different concentrations to improve enzyme production was evaluated. Maximum lipase activity (125.33±2.96 U/mL) as well as maximum biomass production (22.36±0.99 mg/mL) was observed for olive oil when used at a concentration of 2% (v/v) of the basal medium. In the presence of surfactants, the highest lipase activity occurred when SDS and Tween 80 were added at the time of fungal inoculation. SDS proved to be the best surfactant having 110.66±3.52 U/mL lipase activity. The effects of the divalent metal ions (iron and magnesium) on lipase activity were also studied. Iron inhibited, whereas magnesium slightly increased lipase activity. The optimum pH for lipase production was 5.7 while 32°C proved to be the best temperature for lipase production.     

Host plants influence susceptibility of whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) to the entomopathogenic fungus Isaria fumosorosea (Hypocreales: Cordycipitaceae)

We quantified the tritrophic effect of host plant on the susceptibility of the sweetpotato whitefly Bemisia tabaci (Genn.) to a fungal pathogen in the laboratory. Second-instar whiteflies reared on cucumber, eggplant, tomato and bean plants for six generations were exposed to conidial suspensions of Isaria fumosorosea isolate IF-1106. Our results did not detect differences in response (proportional survival or median lethal time, LT₅₀ days) among insect populations derived from different plants that were treated with 10⁷ conidia/ml. However, at concentrations ≤ 5×10⁶ conidia/ml, whiteflies reared on bean and tomato died significantly more quickly (i.e. LT₅₀ of 4–5 days) compared with cucumber and eggplant reared populations (5–7 days). Bean and tomato-reared populations were also more susceptible to mycosis (LC₅₀ ≈ 6 × 10⁵ conidia/ml) compared with those reared on cucumber (1.9 × 10⁶ conidia/ml) and eggplant (1.5 × 10⁶ conidia/ml). A separate study confirmed that this differential response of whitefly populations to I. fumosorosea was not explained by differences in deposition rate of conidia on leaf surfaces (i.e. a dosage effect). Our findings show that host plants affect the pathogenicity and virulence of a herbivore pathogen, but depend on the rate of exposure (inoculum) applied.     

Simplicillium spumae (Cordycipitaceae,Hypocreales), a new hyphomycetes from aquarium foam in Japan

Five Simplicillium isolates were obtained from foam generated in an aquarium in Minato-ku, Tokyo, Japan. Using a combination of micro-morphological characteristics and multigene (ITS, SSU, LSU, TEF) phylogenics, the isolates were identified as S. subtropicum and a new species which were previously undescribed. Simplicillium spumae, which is a new species, differs from known Simplicillium species in the morphology of their conidia and conidiophores.     

Production and regulation of extracellular proteases from the entomopathogenic fungus Isaria fumosoroseus (Cordycipitaceae; Hypocreales) in the presence of diamondback moth,Plutella xylostella cuticle

Extracellular proteases of entomopathogenic fungi have been implicated as components of the insect infection process. To elucidate their role in the infection process, it is essential to characterize these enzymes. During the current studies synthesis and regulation of extracellular proteases (Pr1 and Pr2) by Isaria fumosoroseus (Cordycipitaceae; Hypocreales) isolate IF28.2 were investigated as a function of carbon source (with special reference to diamondback moth cuticle), temperature and pH. The highest level of Pr1 and Pr2 activity were found in the supernatants from 1% glucose plus 1% diamondback moth cuticle (18.83±1.25 and 12.44±1.36 U/mg per h for Pr1 and Pr2, respectively). Maximum Pr1 production by the depressed mycelia was observed from the supernatants having diamondback moth cuticle as a nutrient source, whereas depressed mycelia showed maximum Pr2 activity from the cultures having chitin as the basic nutrient. The optimum pH for Pr1 and Pr2 activity was 8 while 35°C was the best temperature for protease production.     

Effect of photoperiod and host distribution on the horizontal transmission of Isaria fumosorosea (Hypocreales: Cordycipitaceae) in greenhouse whitefly assessed using a novel model bioassay

A model bioassay was used to evaluate the epizootic potential and determine the horizontal transmission efficiency of Isaria fumosorosea Trinidadian strains against Trialeurodes vaporariorum pharate adults under optimum conditions (25±0.5°C, ~100% RH) at two different photoperiods. Untreated pharate adults were arranged on laminated graph paper at different distributions to simulate varying infestation levels on a leaf surface. Four potential hosts were located 7, 14 and 21 mm away from a central sporulating cadaver simulating high, medium and low infestation levels, respectively. Percent hosts colonized were recorded 7, 12, 14 and 21 days post-treatment during a 16- and 24-h photophase. After 21 days, mean percent hosts colonized at the highest, middle and lowest infestation levels were 93 and 100%, 22 and 58%, 25 and 39% under a 16- and 24-h photophase, respectively. From the results, it was concluded that the longer the photophase, the greater the percentage of hosts colonized, and as host distance increased from the central sporulating cadaver, colonization decreased. The use of this novel model bioassay technique is the first attempt to evaluate the epizootic potential and determine the horizontal transmission efficiency of I. fumosorosea Trinidadian strains under optimal environmental conditions at different photoperiods. This bioassay can be used to assess horizontal transmission efficiency for the selection of fungi being considered for commercial biopesticide development.     

Crinipellis pedemontana sp. nov. (Agaricomycetes), a new basidiomycete from Italy

Crinipellis pedemontana, a new agaric growing on stems of dead grasses in an Italian urban park, is described and illustrated. It fits in sect. Grisentinae on account of the green reaction with KOH and ammonia and its bright colored pileus. The unique combination of purple-violaceous tinges in the pileus and the presence of chlamydospores in the pileipellis characterize this species as new. Its taxonomic position within Crinipellis and the related genus Moniliophthora is discussed.     

Heterorhabditidoides chongmingensis gen. nov., sp. nov. (Rhabditida: Rhabditidae), a novel member of the entomopathogenic nematodes

During a recent soil sample survey in Eastern China, a new entomopathogenic nematode species, collected from the Chongming Islands in the southern-eastern area of Shanghai, was discovered. Morphological characteristics of different developmental stages of the nematode combined with molecular data showed that this nematode is a new genus of Rhabditidae, and described as Heterorhabditidoides chongmingensis gen. nov., sp. nov., for that it shares more morphological characteristics with heterorhabditids than with steinernematids. For males, the papillae formula of bursa is 1, 2, 3, 3, with constant papillae number in the terminal group, stoma tubular-shaped and about 1.5 head width; cheilorhabdions cuticularized, esophageal collar present and long, median bulb present. For infective juveniles, EP = 90 (80-105) μm, ES = 104 (92-120) μm, tail length = 111 (89-159) μm, and a = 19.1 (15-21). The percentages of the nucleotides A, T, C and G in the ITS1 regions of the new species are significantly different from those of heterorhabditids and other rhabditids. Molecular phylogenetic trees based on 18S rDNA and the internal transcribed spacer (ITS) sequences data revealed that the new entomopathogenic nematode species forms a monophyletic group, which is a sister group of the clade comprised of some genera of Rhabditidae.     

Steinernema ceratophorum n. sp. (Nematoda: Steinernematidae), a new entomopathogenic nematode from north-east China

Steinernema ceratophorum n. sp. was isolated from soil in Liaoning and Jining Provinces of China. Morphological, cross-breeding and DNA studies support the distinctiveness of S. ceratophorum n. sp. in comparison with four morphologically similar Steinernema species: S. affine, S. intermedium, S. riobrave and S. bicornutum. Diagnostic characters include: body length of infective juvenile, juvenile lip region with two horn-like structures and lateral field with six or eight longitudinal ridges; tail tip of first and second generation males lacking mucron; spicules of first generation male curved, dark yellow in colour and 71 µm long; tail tip of first generation female with small mucronate projection; and second generation female with fine mucronate process about 5.5 µm long. In addition, the restriction fragment length pattern (RFLP) of the ITS region of the ribosomal DNA repeat unit is different from other Steinernema spp., and S. ceratophorum n. sp. did not hybridise with S. riobrave, the species with the most similar RFLP.     

Zygozyma suomiensis,sp. nov. (Lipomycetaceae), a new species from Finland

A new species of the genus Zygozyma, Z. suomiensis, is described, based on the study of a single strain, derived from a bovine skin lesion.     

Debaryomyces udenii,sp. nov. (Saccharomycetaceae), a new species from soil

A new, soil-associated species of the genus Debaryomyces, D. udenii, is described. The species is characterized by pusticulate rather than verrucate ascospores, and slowly lytic asci.     

Effects of the fungus Isaria fumosorosea (Hypocreales: Cordycipitaceae) on reduced feeding and mortality of the Asian citrus psyllid,Diaphorina citri (Hemiptera: Psyllidae)

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is an important citrus pest primarily because it transmits bacteria putatively responsible for huanglongbing, a serious disease of citrus. We present a study on the effects of blastospore and conidial formulations of Isaria fumosorosea Wize on feeding rates and mortality of adult psyllids in laboratory bioassays. Information on quantities of honeydew droplets was used to make inferences on feeding rates. Psyllids treated with the blastospore formulation of I. fumosorosea produced fewer honeydew droplets compared to the conidial treatment and control beginning within the first 24 h after treatment. The highest daily mean number of droplets thereafter never exceeded 2.4 drops compared to 4 and 8 for the conidial treatment and control, respectively. The mean number (±SEM) of honeydew droplets produced per psyllid per day over 7 days was significantly higher in the control (5.5±0.5) compared to the blastospore treatment; however, there were no significant differences between the treatments. Psyllids treated with the conidial formulation of the pathogen showed no significant reduction in feeding activity until 4 days after treatment. One and 2 day's post-exposure, mortality of psyllids in the blastospore treatment ranged from 8 to 25% compared to 0% in the conidial and control treatments. By 7 days post-exposure, psyllid mortality reached 100% under both fungal treatments compared to none in the controls. This study documented that adult psyllids infected by I. fumosorosea (PFR 97) produce less honeydew than healthy psyllids and suggests that they may feed less, which could potentially reduce the spread of huanglongbing.     

Steinernema weiseri n. sp. (Rhabditida,Steinernematidae), a new entomopathogenic nematode from Europe

Steinernema weiseri n. sp. is described from a roadside with apple trees near Ceské Budejovice, Czech Republic. The species is also widely distributed in Germany and Slovakia, from where it had previously been reported as Steinernema spec. F. The British Steinernema sp. D1 is considered conspecific with S. weiseri n. sp. Males of the new species are mainly characterised by light brown, slightly curved spicules with a long manubrium and the presence of a short tail mucron in the second generation. Third-stage infective juveniles are characterised by a `medium size' body and tail length, short hyaline tail portion (mostly around 1/3 of tail length), the excretory pore situated in the mid-pharynx region, lip region slightly offset, angular and flattened, and the lateral field having nine equally developed lines separated by eight distinct ridges. S. weiseri n. sp. is most similar to S. feltiae, with which it did not hybridise. RFLP analysis of the ITS region of the rDNA repeat shows S. weiseri n. sp. to be distinct from 50 other Steinernema species and isolates. The new species was found in a wide range of habitats and is readily maintained on Galleria mellonella larvae.     

Steinernema aciari sp. n. (Nematoda: Steinernematidae), a new entomopathogenic nematode from Guangdong, China

A new species of entomopathogenic nematode, Steinernema aciari sp. n. was described. It was recovered from a soil sample collected from Haimen town, Shantou district in the eastern coast of Guangdong province, the People's Republic of China during a survey for entomopathogenic nematodes. S. aciari sp. n. belongs to the Steinernema glaseri group. It can be separated from all described Steinernema species by the combined morphological and morphometrical characters of various stages of the nematodes. For male, the new species can be recognized by spicule length (86+/-6.3 microm); spicule tip blunt with a hook-like structure; gubernaculum with a short and Y-shaped cuneus and corpus well-separated posteriorly. For infective juvenile, the combination of the following characters: body length (1113+/-68 microm), distance from anterior end to excretory pore (95+/-3.7 microm), tail length (78+/-5.2 microm), and E % (123+/-7) can be used to differentiate the new species from other nematodes. For female, the tail (conoid with a long mamillate terminus and a distinct postanal swelling) and vulva (slightly protruding from body surface with conspicuous double flapped epiptygma) shapes can be used as diagnostic characters for the new species. The new species can also be distinguished from other Steinernema species by DNA sequences of either a partial 28S rDNA or the internal transcribed spacer regions of rDNA, and from the close related species S. glaseri, Steinernema longicaudum CWL05, and Steinernema guangdongense by cross-breeding test.     

Trichosporonoides nigrescens sp. nov., a new xerophilic yeast-like fungus

A new yeast-like fungus isolated from jam on two occasions is described as Trichosporonoides nigrescens. It is a true xerophile, able to grow at water activities at least as low as 0.75. For this reason, its carbon assimilation pattern was examined using both dilute (standard) and concentrated carbon sources: six to nine compounds not assimilated at the standard concentrations of 0.5% (0.001 to 0.1 mol/kg) were assimilated at 1.5 to 2.0 mol/kg. Cell wall and septal ultrastructure, DNA base ratios, and the nuclear cycle were examined to determine the generic and higher taxonomic affinities of this microorganism. All properties were consistent with its assignment to Trichosporonoides Haskins et Spencer. However, while ultrastructure indicated an affinity with the Basidiomycetes, the mitotic nuclear cycle and the xerophilic nature of this fungus suggested an Ascomycete affinity. The GC content (57%) is consistent with assignment to either. No teleomorphic state was found.     

'Sporidesmium' lichenicola sp. nov., a new lichenicolous fungus on Leptogium from Venezuela

'Sporidesmium' lichenicola sp. nov. is described from the decaying thallus of an unidentified Leptogium species growing on unidentified tree bark from the Guaramacal National Park in Boconó, Táchira, in western Venezuela. This is only the second lichenicolous species to be described under this generic name, and differences from that species and similar species in Sporidesmium s. lat. are discussed. A more precise generic placement will have to await a molecularly based taxonomy of the genus. The original material comes from a mycologically little explored region of the country, and brief information on previous mycological (including lichenological) studies in the area is provided for the first time in English. The new combination S. sinense (syn. Repetophragma sinense) also is made.     

The effect of entomopathogenic Lecanicillium spp. (Hypocreales: Cordycipitaceae) on the aphid parasitoid Aphidius colemani (Hymenoptera: Aphidiinae)

The effects of entomopathogenic Lecanicillium spp. (Zare and Gams) on Aphidius colemani (Viereck) adult longevity and ovipositional behavior and on the emergence rate and longevity of the F₁ generation of this parasitoid insect were evaluated under laboratory conditions. Moreover, the ability of three strains of Lecanicillium spp. to penetrate the cuticle of aphids parasitized by A. colemani at different larval stages was investigated. Although fungal treatments at high concentrations (10⁷?conidia/ml) reduced adult longevity, no significant effects were observed at lower concentrations. The number of ovipositional probings, sting failure rate of the ovipositor, and time intervals between periods of ovipositional behavior were not significantly different between the experimental groups and controls at all concentrations for all fungal strains. Fungal inoculation showed no significant effects on the emergence rate and longevity of the adult A. colemani F₁ generation. Fungal penetration was detected in 60?% of aphids inoculated with fungi 7?days after parasitoid exposure. However, fungal detection was drastically reduced and undetectable when fungal inoculation occurred 8 and 9?days after parasitization, respectively. Our results suggest the coapplication of Lecanicillium spp. and A. colemani for aphid control is possible under certain conditions because of their high compatibility.     

Macarthuria intricata sp. nov. (Aizoaceae), a new species from South Western Australia

Macarthuria intricata is described and illustrated. The new species is confined to the Tamala Vegetation System at the northern extremity of the South Western Botanical Province.     

Caecomyces sympodialis sp. nov., a new rumen fungus isolated from Bos indicus

A new anaerobic rumen fungus was isolated from the rumen fluid of a yellow cow (Bos indicus). This fungus appears to be a previously undescribed species of the genus Caecomyces, it possessing uniflagellate zoospores, a spherical holdfast, tubular sporangiophores and bulbous rhizoids. This new fungus also features distinctive multisporangiate thallus sympodially distributed on sporangiophores. The fungus resembles Caecomyces communis and C. equi in that it characterizes bulbous rhizoids and uniflagellate zoospores but differs from C. communis and C. equi in that it possesses multisporangiate and sympodial sporangia. This new fungus and Cyllamyces aberensis both reveal similar morphology during early thallus development in having a spherical holdfast, but they vary from unbranched sporangiophores and additional bulbous rhizoids. In addition, the molecular phylogenetic analyses ITS1 (internal transcribed spacer 1) also conform to the results of the morphological examinations of Caecomyces. For the mentioned reasons, this new species of fungus is described as Caecomyces sympodialis sp. nov. The genera of Neocallimasticaceae and species of Caecomyces are also keyed out.     

Aloe nugalensis sp. nov. (Asphodelaceae), a new gypsum endemic from northeastern Somalia

The new species Aloe nugalensis Thulin, a shrubby plant with long, drooping leaves and a drooping, branched inflorescence with long‐pedicellate orange red flowers, is described from a gypsum hill in the Nugaal valley of northeastern Somalia. The only known material is an individual grown in the Botanical Garden of Uppsala University from seeds collected in Nov 1985 that first flowered in Feb 2011.