Comparative laboratory studies on three fungal pathogens of the elm bark beetle Scolytus scolytus: Pathogenicity of Beauveria bassiana,metarhizium anisopliae, and Paecilomyces farinosus to larvae and adults of S. scolytus

The entomogenous fungi Beauveria bassiana (nine isolates), Metarhizium anisopliae (seven isolates), and Paecilomyces farinosus (four isolates) were tested as pathogens of larvae of the elm bark beetle, Scolytus scolytus. Single isolates of B. bassiana and M. anisopliae were also tested against adult beetles. Of the 21 isolates tested as conidial suspensions against larvae, all proved pathogenic. The three most and least virulent isolates were, respectively, isolates of B. bassiana and M. anisopliae. The other isolates fell between these two extremes, with the four P. farinosus isolates all moderately virulent. Spore retention on larvae following inoculation was estimated by washing conidia off the larvae. From the results it was possible to relate larval mortality to the approximate spore dose causing infection at different spore concentrations. Thus, application of spores of the three pathogens at a concentration of 10³ spores/ml resulted in limited mortality. At this concentration, an average of only a single spore was recovered from the inoculated larva. Adult bark beetles also proved susceptible to infection by isolates of B. bassiana and M. anisopliae. They were exposed to discs of elm bark dipped in a conidial suspension. It was estimated that a dose of less than 100 spores could cause infection of beetles following feeding on the elm bark discs.     

Studies on the impact of two Nosema isolates from Bulgaria on the gypsy moth (Lymantria dispar L.)

We investigated host-parasite interactions of two Nosema-type microsporidian isolates recovered from populations of Lymantria dispar L. in northwestern Bulgaria, one near Veslec and one near Levishte. Bioassay studies produced information on development, stage specific mortality, pupation, and adult eclosion of infected individuals. Horizontal transmission of the two isolates was investigated in a second set of experiments. At dosages ranging from 2 x 10(2) to 5 x 10(4) spores/microl, the infection rates varied between 77 and 100% for the isolate from Veslec and between 92 and 99% for the Levishte isolate. The Veslec isolate caused a slightly higher mortality rate and the median time to death was shorter compared to the isolate from Levishte. The total mortality for both isolates varied between 79 and 99%, independent of spore dosages. A lower relative growth rate was recorded for male and female L. dispar larvae infected with either isolate during the third larval instar and a higher relative growth rate during the fourth instar compared to the control groups. Pupal weight did not differ significantly among females, but male infected pupae were heavier than the controls. Nosema sp. [Veslec] was as efficiently transmitted as Nosema sp. [Levishte]; 42% of the susceptible larvae became infected with the Veslec isolate when uninfected larvae were exposed to infected larvae; 43% of larvae became infected with the Nosema sp. [Levishte]. The latency period varied between 7 and 8 days for both isolates.     

Pathogenicity of fungi isolated from the larvae of the mangrove crab,Scylla serrata, in Indonesia

Heavy mortality reaching almost 100% occurred in the larvae of the mangrove crab,Scylla serrata, from July to December 1997 at the hatchery of Gondol Research Station for Coastal Fisheries in Bali, Indonesia. Mortality was observed in larvae after hatching from the eggs. The affected larvae were whitish and filled with numerous aseptate hyphae. Three fungi belonging to the order Lagenidiales,Lagenidium callinectes, Haliphthoros milfordensis, andHalocrusticida sp., were isolated from the infected larvae. Pathogenicity tests of the infected fungi against the larvae of mangrove crab demonstrated that all isolates were pathogenic.     

Phylogeny of certain biocontrol agents with special reference to nematophagous fungi based on RAPd

A number of phylogenetic studies have been carried out on biocontrol agents having similar biological control activity. However, no work has been carried out to determine the phylogenetic relationship amongst various groups of biological control agents with varied biocontrol properties. Our aim was to derive a phylogenetic relationship between diverse biocontrol agents belonging to the deuteromycetes and determine its correlation with their spore morphology and their biocontrol activity. RAPD was used to assess genomic variability in fungi used as biological control agents which included ten isolates of nematophagous fungi such as Arthrobotrys sp., Duddingtonia sp., Paecilomyces sp. and Verticillium sp., along with two isolates of fungal biocontrol agents such as Trichoderma sp. and two isolates of entomopathogenic fungi including Beauveria sp. A plant pathogenic fungus, Verticillium alboatrum was also included to increase the diversity of Deuteromycetes used. A similarity matrix was created using Jaccard's similarity coefficient & clustering was done using unweighted pair group arithmetic mean method (UPGMA). The final dendogram was created using a combination of two programs, Freetree and TreeExplorer. The phylogenetic tree constructed from the RAPD data showed marked genetic variability among different strains of the same species. The spore morphologies of all these fungi were also studied. The phylogenetic pattern could be correlated with the conidial and conidiophore morphology, a criterion commonly used for the classification of fungi in general and Deuteromycetes in particular. Interestingly, the inferred phylogeny showed no significant grouping based on either their biological control properties or the trapping structures amongst the nematophagous fungi as reported earlier by other workers. The phylogenetic pattern was also similar to the tree obtained by comparing the 18S rRNA sequences from the database. The result clearly indicates that the classical method of classification of these deuteromycete members on the basis of their spore morphology is reliable and could be used for identification of these fungi at species level. The PCR fragment pattern polymorphism exhibited by the various species of a genus and different strains of a species indicates that construction of probes from one or more of these fragments will prove to be useful as a rapid tool for identification of species and strains of nematophagous fungi in future.     

云南香格里拉叉襀科昆虫病原菌的鉴定

叉襀科Nemouroidea是襀翅目中数量最多的一个科,叉襀科昆虫也是水生生态系统环境监测的重要指示生物.为明确云南香格里拉叉襀科稚虫自然病死虫体上的病原菌并研究该病原菌对叉襀科稚虫的致病性,本研究利用常规组织分离法分离云南香格里拉叉襀科稚虫病原菌,结合形态学与分子生物学方法对两株病原菌进行鉴定,利用柯赫法则及二项式回归模型分析病原菌致病性.结果表明,菌株PN1为细囊霉菌Leptolegria sp.,菌株PN2为冻土毛霉Mucor hiemalis;PN1及PN2处理组对叉襀科稚虫的致病性稍弱,其中PN1处理组的致病性低于PN2处理组,PN2处理组的致病性与对照组无明显差异.综上所述,本研究首次在襀翅目昆虫中报道了细囊霉菌Leptolegnia sp.和冻土毛霉M．hiemalis,两株病原真菌均属于水生弱寄生菌,在稚虫处于亚健康状态及抵抗力降低时易感,病原菌加速寄生过程使稚虫死亡.     

Fluctuations in haemocyte density and microbial load may be used as indicators of fungal pathogenicity in larvae of Galleria mellonella

A positive correlation exists between the pathogenicity of bacteria and fungi when evaluated in the insect Galleria mellonella and mice. This work sought to determine whether fluctuations in the number of haemocytes and the proliferation of yeast cells in infected larvae could be used to determine the relative pathogenicity of a range of yeast isolates. Larvae were inoculated with 1 x 10(6) stationary-phase yeast cells and incubated in the dark at 30 degrees C for 48 h. The results indicated that larvae inoculated with the most pathogenic isolates (i.e. those capable of killing >80% of infected larvae) showed a significant reduction in haemocyte density. Larvae inoculated with isolates of low pathogenicity (i.e. capable of killing <20% of infected larvae) demonstrated only a small fluctuation in haemocyte numbers. The most pathogenic yeast isolates proliferated in the larvae, whereas the isolates of low pathogenicity did not. These results demonstrate a relationship between the ability of yeast isolates to kill larvae and changes in haemocyte density and yeast cell density in infected larvae. These end points may extend the applicability of the G. mellonella system for use with a wider range of microbial isolates.     

Selection of a Highly Virulent Fungal Isolate,Metarhizium anisopliae Ma TB 101, for Control of Taro Beetle,Papuana uninodis (Coleoptera: Scarabaeidae)

Fungal isolates (31 Metarhizium anisopliae var. anisopliae , five M. anisopliae var majus , three Beauveria bassiana and four B. brongniartii ) originating from a wide range of geographical locations, insect species and soil types were tested against Papuana uninodis (Coleoptera: Scarabaeidae). In a first test series, spores were applied topically to third-instar larvae and adults. The most effective strain against P. uninodis larvae and adults was Ma TB 101, a M. anisopliae isolate from adult P. woodlarkiana . For adults, strain Ma FI 384, a M. anisopliae from Popillia japonica , was almost equally effective. The 11 most effective isolates (nine M. anisopliae var. anisopliae , one B. brongniartii and one B. bassiana ) with LT values of less than 6 weeks in 50 adults and/or less than 4 weeks in larvae were tested for their efficacy against adults and larvae of P. uninodis by application of spores to soil (107 spores/g). Ma TB 101 was significantly more effective against both adults and larvae (LT ca. 10 days) than all other isolates (LT > 50 50 3 weeks). Two other M. anisopliae strains, Ma F 248 from soil and Ma FI 384 from P.japonica , were more effective than most isolates in adults. The latter three M. anisopliae isolates were tested in a concentration series against third-instar larvae and adults. Mortality was concentration related. Isolates Ma F 248 and Ma FI 384 did not achieve 50% mortality within the test period at concentrations lower than 107 spores/g of soil or feed. For concentrations of Ma TB 101 from 1 107 to 2 105 spores/g the LT ranged from 13 to 30 days in adults and 12 to 24 50 days in third-instar larvae. Accordingly, concentrations causing 50% mortality (LC ) for Ma 50 TB 101 were significantly lower than for the two other M. anisopliae isolates tested.     

Natural products of Alternaria sp., an endophytic fungus isolated from Salvadora persica from Saudi Arabia

This study is to evaluate the potential of endophytic fungi of Salvadora persica for the production of bioactive compounds against pathogenic bacteria and fungi. Forty-two fungal isolates were obtained from 135 young and old stem and 125 root segments. Those 42 isolates representing ten fungi include: Trichoderma sp. (the most common), two species of Alternaria, Rhizopus arrhizus and 6 sterile mycelia. The ten fungi were grown in liquid culture and their crude extracts were tested against pathogenic bacteria and fungi. Nine crude extracts gave positive reactions against pathogenic bacteria of which Alternaria sp. (A8) was chosen further study. The fungal isolate was growing as sterile mycelium and was identified by phylogenetic analyses based on LSU rDNA sequence data and it might represent undescribed species of Alternaria. Sixty-two bioactive chemical compounds were identified from the ethyl acetate crude extracts of Alternaria sp., of which the following were recorded as major compounds in the active sub-fractions. These compounds showed strong antibacterial activity in combination.     

Ultrastructural study of galacturonic acid distribution in some pathogenic fungi using gold-complexed Aplysia depilans gonad lectin

Aplysia gonad lectin, isolated from the mollusc Aplysia depilans, was successfully conjugated to colloidal gold and used for ultrastructural detection of galacturonic acids in some pathogenic fungi. These sugar residues were found to occur in the fibrillar sheath surrounding hyphal cells of Ascocalyx abietina and in intravacuolar dense inclusions of this fungus spores. In hyphae and spores of Ophiostoma ulmi, galacturonic acids were detected mainly in the outermost wall layers. In contrast, these saccharides appeared associated with the innermost wall layers and especially the plasma membrane of Verticillium albo-atrum cells. Galacturonic acids were found to be absent in cells of Fusarium oxysporum f.sp. radicis-lycopersici and Candida albicans. These cytochemical data indicate therefore that a heterogeneity in wall composition exists between ascomycete fungi. The significance of the presence of galacturonic acids in the cell walls of certain fungi is still open to question.     

Selection of isolates of entomopathogenic fungi for controlling Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) and their compatibility with insecticides used in tomato crop

The activity of Metarhizium anisopliae and Beauveria bassiana towards eggs and larvae of Tuta absoluta (Meyrick) was evaluated. Our data showed that the isolates were pathogenic to both developmental stages tested and the eggs were more susceptible than the 1st instars. The isolates URPE-6 and URPE-19 of M. anisopliae were more pathogenic to eggs and larvae, respectively. The compatibility of these two isolates with the insecticides chlorfenapyr, spinosad, indoxacarb, abamectin, and neem were evaluated. Spinosad and indoxacarb were compatible with the two M. anisopliae isolates in all tested concentrations. At the average recommended concentration, chlorfenapyr was compatible to URPE-6 and abamectin to UFPE-19. The use of entomopathogenic fungi associated with compatible insecticides may be a useful alternative to control T. absoluta.     

Studies on bacterial flora in the population of the fall webworm, Hyphantria cunea Drury. (Lep., Arctiidae)

Abstract:  In this study, the bacterial flora of Hyphantria cunea Drury. (Lep., Arctiidae) were investigated during three hazelnut seasons from 1998 to 2000. Four different bacteria were found in dead and living larvae. They were isolated and identified as Bacillus thuringiensis , Escherichia freundii , Micrococcus sp. and Streptococcus sp. Laboratory experiments carried out to determine the insecticidal activities of these isolates showed that E. freundii and Micrococcus sp. did not have any insecticidal effect on second – third instar larvae of H. cunea . However, B. thuringiensis and Streptococcus sp. had 56 and 38% effects, respectively. Crystals and spores from B. thuringiensis were also purified and the crystals, spores and crystals–spore mixture were tested separately against the larvae of H. cunea . It was found that the insecticidal activities of the crystals, spores and crystal–spore mixture were 37.5, 25 and 62.5%, respectively, on second – third instar larvae of H. cunea . These results indicate that the crystal–spore mixture has 6.5% more insecticidal effect than that of the vegetative cells of the B. thuringiensis isolate.     

In vitro interaction of Brazilian strains of the nematode-trapping fungi Arthrobotrys spp. on Panagrellus sp. and Cooperia punctata

In vitro tests were carried out to verify the activity of 26 Brazilian isolates of predatory fungi of the genus Arthrobotrys on a free-living nematode (Panagrellus sp.) and on infective larvae of Cooperia punctata, a parasitic gastrointestinal nematode of cattle. The results showed that the free-living nematode Panagrellus sp. was the most preyed upon, compared to C. punctata, for all the fungal treatments. Also, variable predatory capacity was observed for different fungal isolates belonging to the same genus when applied to different nematode species.     

Potential of Cactoblastis cactorum as a Vector for Fungi Pathogenic to Pricklypear, Opuntia inermis

In Australia, fungi associated with larvae of the biological control agent Cactoblastis cactorum may contribute to the control of the exotic weed pricklypear (Opuntia inermis). C. cactorum larvae were assessed for their ability to vector pathogenic fungi into O. inermis by the infestation of larvae with fungal suspensions. Six fungal isolates caused disease after being carried into the host on external surfaces of larvae, and propagules of one isolate (UQ5109) initiated disease after being transferred from the cladode epidermis into the host by larvae feeding on the plant. Scanning electron microscopy revealed extensive hyphal growth on the external surfaces of larvae infested with several of the isolates. Fungi isolated from field-grown O. inermis cladodes were tested for pathogenicity to this plant in an in vivo plant assay. In total, 152 isolates were screened, 22 of which infected the host in pathogenicity tests. Only 1 (UQ5115) infected undamaged host tissue, whereas the remainder required the host to be wounded before infection could proceed. The majority of isolates were only weakly pathogenic, even when inoculated via wounds, suggesting that most were either saprophytes or weak parasites. This study demonstrates that it is possible for larvae of C. cactorum to transmit fungal pathogens into O. inermis tissue and it has provided a sound basis for future field work to determine the contribution that fungi make to the control of O. inermis.     

Pathogenicity of Beauveria bassiana,Metarhizium anisopliae and Serratia marcescens to the banana weevil Cosmopolites sordidus

Two exotic fungal isolates, one of Beauveria bassiana (268–86) and another of Metarhizium anisopliae (100–82), three local isolates of B. bassiana (isolates I, II, III) and one of the entomogenous bacteria Serratia marcescens, were tested for pathogenicity against the banana weevil Cosmopolites sordidus. All four isolates of B. bassiana and the one of M. anisopliae were found to be pathogenic to third—instar larvae of C. sordidus, causing mortalities of 98–100% by 9 days post—exposure to dry fungal spores. M. anisopliae was the least pathogenic to larvae with LT₅₀ of 4.2 days, compared to 3.5, 3.3, 3.6 and 4.0 respectively for isolates I, II, III and 268–86. B. bassiana was also pathogenic to adult C. sordidus, causing mortalities varying from 63–97% by 35 days post—exposure depending on isolate. As for larvae M. anisopliae exhibited low pathogenicity for the adult C. sordidus. In general, all the fungi tested were less pathogenic to adult weevils (LT₅₀ = 17.5; 12.5; 8.0 and 22.0 days) for isolates I, II, III and 268–86 respectively, while isolate 100–82 failed to kill 50% of adults even by 35 days post—exposure. Incubation of dead weevils in a moist environment led to development of surface mycelia starting from intersegmental junctions. Histopathology revealed extensive destruction of internal organs by hyphae which invaded most of the organs. The LT₅₀ for S. marcescens against C. sordidus larvae was 2.8 days. However, the bacterium did not kill adult C. sordidus even at 10 times the concentration applied on larvae.     

抗多重耐药病原菌的越南槐内生菌TRPH-35的抗菌活性及其鉴定

以大肠杆ATCC25922、金黄色葡萄球菌ATCC25923及其临床多重耐药分离株ETEC-AD9、MRSA-DF12共4株人体病原细菌为靶标菌,采用琼脂块法从越南槐内生真菌中筛选抗菌活性菌株,并以改良的微量肉汤稀释法测定其代谢产物的最小抑菌浓度;根据形态和分子特征鉴定拮抗菌株的分类学地位。结果表明,菌株TRPH-35的活菌块对各病原细菌的抑菌圈直径与阳性对照相当;其代谢产物对各病原菌的最小抑菌浓度为20~40μg/mL;初步鉴定该菌株为刺盘孢。本研究结果表明,菌株TRPH-35鉴定为刺盘孢,对参试病原细菌均显示强的抗菌活性,具有较大的开发应用潜力。     

In vitro activity of Brazilian strains of the predatory fungi Arthrobotrys spp. on free-living nematodes and infective larvae of Haemonchus placei

In vitro tests were carried out to assess the activity of 26 Brazilian isolates of predatory fungi of the genus Arthrobotrys on a free-living nematode (Panagrellus sp.) and on infective larvae of Haemonchus placei, a parasitic gastrointestinal nematode of cattle. The results showed that the free-living nematode Panagrellus sp. was the most preyed upon, compared to H. placei, for all the fungal treatments. Also, variable predatory capacity was observed for different fungal isolates belonging to the same genus when applied to different nematode species.     

Distribution and characterisation of entomopathogenic fungi from Korean soils

We investigated the occurrence of entomopathogenic fungi in 1080 soil samples representing multiple locations and conditions in Korea. Entomopathogenic fungi were isolated from soils using a selective medium containing dodine and antibiotics. Following an initial identification based on morphology, the fungal isolates were more precisely identified by the sequence of their nuclear ribosomal RNA (rRNA) internal transcribed spacer (ITS) regions. As a result, entomopathogenic fungi were found to occur in 32% (342 isolates) of the soil samples studied. The most abundant species were Beauveria spp. (125 isolates) and Metarhizium spp. (82 isolates). Entomopathogenic fungi were more often recovered from natural mountain and riparian soils than from agricultural habitats. The pathogenicity of isolated fungi was evaluated by using wax moth Galleria mellonella L. (Lepidoptera: Pyralidae) larvae. It was determined that 60% (207 isolates) of the isolates were pathogenic using this model. These entomopathogenic fungi may, therefore, have potential use against a variety of agricultural pests. This is the first study of the isolation and distribution of entomopathogenic fungi in representative sampling locations throughout Korea.     

Inhibition of common fouling organisms in mariculture by epiphytic bacteria from the surfaces of seaweeds and invertebrates

A total of 319 bacterial strains isolated from the surfaces of seaweeds and invertebrates were tested for their effects on settlement of Ulvalactuca spores and Hydroidesezoensis larvae in laboratory bioassays. Of the 192 bacterial strains isolated from the surfaces of seaweeds 63 isolates were shown to be inhibitory against the settlement of algal spores and 62 isolates were inhibitory against larval settlement. Thirty-seven percent of the 127 bacterial strains isolated from the surfaces of marine invertebrates were shown to be inhibitory against algal spores and larval settlement. The strain CI4 isolated from an adult Cionaintestinalis showed the strongest inhibitory effect and was identified as Pseudoalteromonas sp. via 16S rRNA gene sequencing. The high proportions of host associated bacteria producing antifouling compounds suggest that these bacteria may help the host organism in the defense against fouling.     

Inhibition of common fouling organisms in mariculture by epiphytic bacteria from the surfaces of seaweeds and invertebrates

A total of 319 bacterial strains isolated from the surfaces of seaweeds and invertebrates were tested for their effects on settlement of Ulvalactuca spores and Hydroidesezoensis larvae in laboratory bioassays. Of the 192 bacterial strains isolated from the surfaces of seaweeds 63 isolates were shown to be inhibitory against the settlement of algal spores and 62 isolates were inhibitory against larval settlement. Thirty-seven percent of the 127 bacterial strains isolated from the surfaces of marine invertebrates were shown to be inhibitory against algal spores and larval settlement. The strain CI4 isolated from an adult Cionaintestinalis showed the strongest inhibitory effect and was identified as Pseudoalteromonas sp. via 16S rRNA gene sequencing. The high proportions of host associated bacteria producing antifouling compounds suggest that these bacteria may help the host organism in the defense against fouling.