Morphological and molecular characterisation of Pythium species causing chilli (Capsicum annuum L.) damping-off

Twenty-five Pythium isolates comprising five species viz., Pythium aphanidermatum, P. deliense, P. graminicola, P. heterothallicum and P. ultimum from different geographical locations of Tamil Nadu (Coimbatore, 4; Cuddalore, 6; Dindigul, 1; Dharmapuri, 1; Erode, 1; Madurai, 1; Namakkal, 7; Thanjavur, 1; Theni, 1; Thirunelveli, 1 and Vellore, 1) isolated from chilli crop were analysed with randomly amplified polymorphic DNA (RAPD) markers. Morphological and molecular characteristics of these different species were correlated with the RAPD. Polymerase chain reaction amplification of total genomic DNA with six random primers generated unique banding patterns depending on the primer and the isolate. The isolate I₁₇ produced identical banding patterns, while other isolates produced dissimilar bands within the particular species, indicating the genetic diversity among the isolates within a species. Morphological characters were also different from each other even in isolate I₁₇ which shared identical bands. Cluster analysis showed minimum and maximum per cent similarities among the tested Pythium species which ranged from 49 to 89%, respectively. RAPD markers were better suited for differentiating isolates within a species rather than species.     

Insecticidal efficacy of two isolates of Beauveria bassiana (Bals.) (Vuill.), on the second larval stage of Leptinotarsa decemlineata (Say) (Col.: Chrysomelidae)

Leptinotarsa decemlineata is a serious potato pest throughout the world that has developed resistance to many insecticides. This study investigated the virulence of two indigenous isolates of Beauveria bassiana, consisting of DEBI007 and IR1217C. Five different concentrations of each isolate, 1 × 10⁷, 3.1 × 10⁷, 1 × 10⁸, 3.1 × 10⁸ and 1 × 10⁹ conidia ml^?1, were applied in bioassay using dipping method on second instar larvae. Control insects were treated with distilled water containing 0.01% Tween-80 (Sigma, Germany) as surfactant. The results showed that mortality percentages due to DEBI007 and IR1217C isolates in the highest concentration were 60% and 57.77%, respectively. There was a significant difference between efficacies of different conidial concentrations; therefore, with rise in conidial concentration, the mortality percentage was increased. The DEBI007 isolate that was previously isolated from the soil was more effective than IR1217C isolate. Calculated LC₅₀ for this effective isolate was 1.4 × 10⁷ conidia ml^?1, which, respectively, is the highest concentration.     

Phylogenetic affinity of Mycochaetophora gentianae, the causal fungus of brown leaf spot on gentian (Gentiana triflora), to Pseudocercosporella-like hyphomycetes in Helotiales

Mycochaetophora gentianae, the causal agent of brown leaf spot on gentian (Gentiana scabra), is characterized by its hyaline besom-like sporophore, although its conidiogenesis and phylogenetic position have so far remained unknown. We isolated the causal fungus from a new host, G. triflora, in Iwate, Japan. Both the G. triflora isolate and the ex-type M. gentianae isolate produced symptoms on G. triflora but not on G. scabra. Microscopic observations of the diseased leaves indicated that conidiogenesis was blastic from short conidiophores, and schizolytic secession of conidia left unthickened and inconspicuous conidial scars on the conidiogenous cells. Conidia were catenate, in branched acropetalous chains; secondary conidia were blastically produced from the first or second cell at the base of primary conidium. The G. triflora isolate was identified as M. gentianae because of its identity to the ex-type in characteristics of culture, pathogenicity, and conidia. Phylogenetic analyses using three ribosomal DNA (rDNA) sequences combined [small subunit (SSU) + large subunit (LSU) + 5.8S rDNA] indicated that both isolates clustered with Rhexocercosporidium carotae, and the cluster was placed within Helotiales–Rhytismatales. Additional analyses using internal transcribed spacers including 5.8S rDNA sequences revealed that both isolates were monophyletic and that they were closely related to three helotialean Pseudocercosporella-like hyphomycetous genera: Helgardia, Rhexocercosporidium and Rhynchosporium.     

Downstream processing of Beauveria bassiana and Metarhizium anisopliae-based fungal biopesticides against Riptortus pedestris: solid culture and delivery of conidia

We established a fungal production platform by focusing on substrates of solid culture for conidial productivity and thermotolerance, and focusing on surfactants to effectively deliver fungal conidia to the Riptortus pedestris cuticles. First, to produce thermotolerant fungal conidia, 2 of each Beauveria bassiana and Metarhizium anisopliae isolates were cultured on 13 cereal substrates for 10 days. Overall, five substrates (millet, non-glutinous Italian foxtail millet, barley, glutinous Italian foxtail millet, and brown rice) produced greatest number of conidia with thermotolerant conidia. When the selected substrates were mixed with minerals, zeolite, perlite or vermiculite to reduce the amount of cereal grains, vermiculite combination showed relatively high conidial production compared to the other mineral combinations. Next, to efficiently deliver the fungal conidia to the cuticles of R. pedestris, six surfactants, CO-2.5, CO-12, LE-7, PE-61, TED-3, and siloxane were each combined with the fungal conidia. The 0.01% combination showed significantly increased insect mortality, which varied depending on isolate. Virulence tests against R. pedestris were performed with conidial suspensions of isolates to assess their virulence. As a result, isolates showed the highest virulence when a virulence test was conducted at 25°C, rather than 20°C, 30°C and 35°C. This work suggests that the combination of cereal grain substrates and vermiculite could be considered for economic conidial production with high thermotolerance, and the CO-12 surfactant is the most suitable for effective delivery to target insects, followed by the information on optimal temperature for virulence against R. pedestris.     

Characterisation of Isaria fumosorosea isolates and their virulence toward the Diamondback Moth,Plutella xylostella

Some morphological and physiological characteristics of an Isaria fumosorosea isolate with diminished virulence, IFCF01-D, and its parent isolate, IFCF01, were evaluated and laboratory bioassays were performed to assess their virulence against Plutella xylostella. The relationship among these traits and virulence against P. xylostella is discussed. There were no significant differences in conidial viability, spore production and the time required for 50% germination (GT₅₀). Spore viability after incubation for 24 h at 25°C was greater than 98% for both isolates tested. Spore production on potato dextrose agar after 14 days incubation at 25°C was 4.68 × 10⁸ and 4.59 × 10⁸ conidia/mL for IFCF01 and IFCF01-D, respectively. When exposed to high temperatures (40, 45, 50 or 55°C) through a water bath for 10 min, conidial germination ranged from 0.83% to 84.0% for IFCF01 and 0% to 86% for IFCF01-D. Germination rate showed a negative relationship with the exposure temperature for both isolates. The per cent germination of isolate IFCF01 24 h after ultraviolet (UV) radiation (18 W, 240–260 nm) varied from 0% to 92% and 0% to 81% for IFCF01-D. Germination rate and the exposure time exhibited a negative correlation for both isolates tested. Conidial surface hydrophobicity of IFCF01 (60%) was significantly higher than that of isolate IFCF01-D (53%). Subsequently, using the cicada exuviae as the substrate for enzymatic analysis, Pr1 and chitinase activity demonstrated the contrasting virulence traits: higher specific activities for the more virulent IFCF01 and lower enzymatic levels for isolate IFCF01-D.     

DNA fingerprinting of Indian isolates of Xanthomonas oryzae pv. oryzae

 A high level of genetic polymorphism was detected among Indian isolates of Xanthomonas oryzae pv. oryzae using hypervariable probes such as a microsatellite oligonucleotide, probe (TG)₁₀, a human minisatellite probe, pV47, an avirulence gene probe, avrXa10 and a repeat clone, pBS101. These DNA probes detected multiple loci in the bacterial genome generating complex DNA fingerprints and differentiated all of the bacterial isolates. Analysis of fingerprints indicated that pV47, (TG)₁₀ and pBS101 have a lower probability of identical match than avrXa10 and therefore are potential probes for DNA fingerprinting and variability analysis of Xanthomonas oryzae pv. oryzae pathogen populations. Cluster analysis based on hybridization patterns using all of the above probes showed five groups at 56% similarity. Studies on the methylation patterns of isolates representing the three important races of X. oryzae pv. oryzae indicated more methylation in the most virulent isolate, suggesting a possible role of methylation in pathogenicity. Received: 8 December 1996 / Accepted: 20 December 1996     

Characterization and virulence of <Emphasis Type="Italic">Lecanicillium lecanii</Emphasis> against different aphid species

Seven isolates of Lecanicillium lecanii (Zimmermann) Zare &; Gams isolated in Spain from infected aphids were characterized using sequences of the Internal Transcribed Spacer (ITS) regions and also based on morphological and physiological characteristics. Four of these seven L. lecanii isolates were selected to assess their virulence against nymphs of Myzus persicae (Sulzer), Nasonovia ribisnigri (Mosley), Macrosiphum euphorbiae (Thomas) and Aphis gossypii Glover. Mortality (%), lethal concentration 50 (LC₅₀) and lethal time 50 (TC₅₀) were calculated. The analysis of the sequences of ITS region confirmed that the new isolates were clearly Lecanicillium lecanii. The set of isolates had similar radial growth (51.5–54.0 mm), except for ICAL1 (39 mm). The germination time 50 (GT₅₀) varied between 10.7 h (ICAL3) and 13 h (ICAL5). The isolate ICAL6 showed the highest value for conidial production (3.4 × 10⁸ con ml^?1) and also produced the highest mortality for M. persicae (95%) and was more virulent than the commercial product Vertalec^® (91.6%).     

Evaluation of Beauveria bassiana (Hyphomycetes) isolates as potential agents for control of Dendroctonus valens

Abstract The red turpentine beetle (RTB), Dendroctonus valens LeConte, as a destructive invasive pest, has become one of the most economically important forest pest in China. Effective control measures are desperately needed. Entomopathogenic fungi, such as Beauveria bassiana, have shown great potential for the management of some bark beetle species. In this study, 12 isolates of B. bassiana from bark beetle were examined for biological characteristics and virulence, to assess their potential as biocontrol agents for RTB. There were significant differences (at P= 0.05) in colony growth rate, conidial yield, conidial germination, tolerance to UV light and extracellular proteases activity among the tested B. bassiana isolates. Isolates, including Bb1801, Bb1906, Bb789 and Bb773, exhibited the best characteristics, because they have faster hyphal growth rate, higher spore production and faster spore germination, higher UV tolerance and protease (Pr1) production. The results of a pathogenicity test of B. bassiana on RTB larvae showed that most isolates of B. bassiana have demonstrated high efficacy and the highest virulent isolate was Bb1801, which killed 100% of the treated insects and had a median lethal time (LT₅₀) of 4.60 days at a concentration of 1×10⁷ conidia/mL. Therefore, isolate Bb1801 has a great potential for sustainable control of RTB in the forest. The correlation between biological characteristics and virulence of the fungal isolates is discussed and the possibility of combination of entomopathogenic fungi with semiochemicals, as one of the promising strategy for RTB control, is considered.     

Evaluation of twenty-eight strains of Heterorhabditis bacteriophora isolated in Azores for biocontrol of the armyworm, Pseudaletia unipuncta (Lepidoptera: Noctuidae)

The armyworm, Pseudaletia unipuncta, is the most important pest in Azorean pastures. Although this pest has some parasitoids and pathogens, additional biological control agents are needed to manage it. Entomopathogenic nematodes, particularly Heterorhaditis bacteriophora, are good candidates because they have been isolated from pastures and crops in almost all islands of the Azorean Archipelago. We tested 28 Azorean isolates of H. bacteriophora in the laboratory against the 6th instar P. unipuncta to determine mortality rates and virulence of each isolate. Plot tests in the field were also conducted to evaluate the best time for application of the selected isolate. All isolates killed the larvae although important differences in the mortality rates were observed. Forty-eight h post exposure (HPE) to the nematode infective juvenile (IJ), insect mortality ranged from 0 to 92.5% and at 96 HPE, mortality ranged from 32.5 to 100%. Based on LC₅₀ and LT₅₀, H. bacteriophora Az29 was the most pathogenic and the remaining 27 isolates were grouped in three classes of virulence. The most virulent class included four isolates with LC₅₀ ranging from 180 to 327 IJs/insect and LT₅₀ from 44 to 62.9 h. These isolates were obtained from three of the nine islands. High intrapopulational variability was detected on isolates in the moderately virulent class suggesting that these isolates are good candidates for genetic improvement. Field tests showed H. bacteriophora Az29 was more effective to control P. unipuncta larvae than Steinernema carpocapsae Az20 and H. bacteriophora Az32, belonging to the less virulent class. These tests also showed that applications performed during May resulted in better control than in July.     

Induction of systemic resistance in rice to bacterial blight by 1,2,3-benzothiadiazole 7-carbothioic acid-S-methyl ester (BTH) treatments

Use of BTH to evaluate the disease severity and induction of systemic resistance in rice to bacterial blight caused by Xanthomonas oryzae pv. oryzae is investigated. A new batch of 25 isolates of Xanthomonas oryzae pv. oryzae was obtained from infected rice lead tissues collected from Pattambi, Kerala, south India. Their identification was confirmed by the plant inoculation test on to IR24 rice plants which produced characteristic bacterial blight lesions. Among the 25 of X.o. pv. oryzae, four of the isolates were also virulent to IRBB21 rice plants (a near isogenic line of IR24) which carry the Xa-21 gene for BB resistance. The results confirm that there are pathogen strains in India which can overcome Xa-21. Development of BB lesions developed in IR24 (BB susceptible) plants after they were treated with BTH applications either as seed treatment or as foliar spray at 0.1, 0.5, 0.1 and 2.0 mM concentrations showed that even at 2.0 mM concentrations, IR24 plants were still susceptible to the pathogen. There was very little or marginal effect of BTH on the induction of resistance to BB in IR24 rice plants. When the same concentrations of BTH were applied to IRBB21 (Xa-21) rice plants, they showed pronounced triggering of systemic resistance to BB pathogen even at 0.1 mM concentration of BTH applied either as seed treatment or as foliar spry. Disease severity index was reduced to 5 (against a score of 9 in untreated) and there was 85–86% reduction in BB incidence in plants that received 0.1 mM BTH. These results provide evidence that BTH-induced systemic resistance complements the R-gene resistance in IRBB21 plants but not in IR24 rice plants.     

Effect of azoxystrobin and kresoxim‐methyl on rice blast and rice grain yield in China

Sensitivity to azoxystrobin and kresoxim‐methyl of 80 single‐spore isolates of Magnaporthe oryzae was determined. The EC₅₀ values for azoxystrobin and kresoxim‐methyl in inhibiting mycelial growth of the 80 M. oryzae isolates were 0.006–0.056 and 0.024–0.287 µg mL^?1, respectively. The EC₅₀ values for azoxystrobin and kresoxim‐methyl in inhibiting conidial germination of the M. oryzae populations were 0.004–0.051 and 0.012–0.105 µg mL^?1, respectively. There was significant difference in sensitivity to azoxystrobin or kresoxim‐methyl between the tested isolates representing differential sensitivity to carbendazim (MBC) and kitazin P (IBP); however, there was no correlation between this difference in sensitivity to azoxystrobin or kresoxim‐methyl and sensitivity to MBC or IBP, indicating that there was no cross‐resistance between azoxystrobin or kresoxim‐methyl and MBC or IBP. In the protective and curative experiments, kresoxim‐methyl exhibited higher protective and curative activity than azoxystrobin when applied at 150 and 250 µg mL^?1 accordingly, while azoxystrobin exhibited stronger inhibitory activity against M. oryzae isolates than that of kresoxim‐methyl in the in vitro test. The results of field experiments also suggested that both azoxystrobin and kresoxim‐methyl at 187.5 g.a.i. ha^?1 gave over 73% control efficacy in both sites, exhibiting excellent activity against rice blast. Taken together, azoxystrobin and kresoxim‐methyl could be a good substitute for MBC or IBP for controlling rice blast in China, but should be carefully used as they were both at‐risk.     

Potential of Metarhizium anisopliae and Beauveria bassiana to control Dinoderus porcellus (Coleoptera: Bostrychidae) infesting yam chips

The beetle Dinoderus porcellus Lesne is a serious storage insect pest that causes important losses by destroying stocks of yam chips. In the aim to found an alternative control method to the use of synthetic insecticides for its management, the virulence of the entomopathogenic fungi Beauveria bassiana (Balsamo) Vuillemin (isolate Bb115) and Metarhizium anisopliae (Metschnikoff) Sorokin (isolate Met 31) against adults of D. porcellus was evaluated under laboratory conditions (25 ± 2 °C and 70 ± 5% RH). Then, the effectiveness of the most virulent entomopathogenic fungus as biological agent against D. porcellus was assessed under farmer storage conditions. For each entomopathogenic fungus isolate, four conidial concentration (0, 10⁵, 10⁷, and 10⁹ conidia/mL) at the dose of 1 µL were inoculated topically on D. porcellus adults (3–5 days old). Observations focused on insect mortality, cadaver sporulation and weight loss of yam chips. Lethal dose and lethal time values were estimated using probit analysis. Both fungal isolates at all conidial dose caused more than 50% mortality on day 7, with the highest mortality (94.44%) achieved using B. bassiana at the 10⁹ conidia/mL. LT₅₀ values for B. bassiana and M. anisopliae isolates were 2.63 and 3.35 days, respectively, while their LT₉₀ values were 6.15 and 9.87 days, respectively. Yielding the lower LD₉₀ values and the highest rates of cadaver sporulation, B. bassiana isolate appeared as the most virulent against D. porcellus. After 3 months of storage, comparatively to the control, the B. bassiana isolate at the highest conidial dose (10⁹ conidia/mL) significantly reduced D. porcellus populations, and weight loss of yam chips. This study revealed the potential of B. bassiana and M. anisoplae isolates as biological control agent against D. porcellus for yam chips protection.     

Virulence potential of some fungal isolates and their control-promise against the Egyptian cotton leaf worm,Spodoptera littoralis

A preliminary virulence test of four fungal isolates, Beauveria bassiana IMI 382302, Beauveria bassiana IMI 386701, Trichoderma harzianum T24 and Aspergillus flavus Link against larvae of Spodoptera littoralis was performed. The most effective isolates against larvae of S. littoralis were B. bassiana 302 and T. harzianum T24, which also showed the lower percentage of pupation compared with the other two isolates under the same conditions of treatments. Three concentrations (1 × 10⁶, 1 × 10⁷ and 1 × 10⁸ ml^?1) of the aqueous conidial suspension of the four tested isolates were carried out against both larval and pupal stages of S. littoralis within five days post-treatment. T. harzianum T24 showed 80% larval mortality only when applied at the highest conidial concentration, while A. flavus showed 100% pupal mortality only, at all of its conidial concentrations. However, B. bassiana IMI 382302 showed relatively high dose-dependant larval and pupal mortalities, while strain IMI 386701 of B. bassiana showed a very weak mortality against pupae at higher concentrations, and no virulence against larvae was recorded. Enzymatic and antibiosis bioassays of the four fungal isolates showed relatively high activities against Fusarium spp. for most of the tested isolates. Clear zone of enzyme activity on agar plates proportionally increased with increasing the concentration of enzyme substrate and prolongation of the incubation period. Mtabolites produced in the agar culture inhibited the growth of Fusarium spp. and the productivity differed greatly among isolates or strains of the same isolate. Volatile and non-volatile compounds produced by A. flavus Link showed a higher inhibition activity against Fusarium spp. compared with the other fungal isolates. The humoral antifungal response of insect host is relatively high compared to the anti-bacterial one. Injection of larvae with the immune sensitive bacteria Micrococcus luteus (5 × 10³ bacteria/larva) showed a detectable humoral response by 2 h, peaked around 12 h and became hardly detectable by 24 h post-injection. Injection of larvae with conidial suspension (5 × 10³ conidia/larva) from each of the fungal isolates showed humoral antifungal activity against B. bassiana IMI 386701 and A. flavus only. This activity was detectable by 12 h, peaked around 36 h and became hardly detectable by 48 h post-injection. Although the humoral antifungal response was started slowly compared to the antibacterial one, it lasted for longer and enabled larvae to withstand the infection with these immune-sensitive fungal strains. No humoral activity was detected against B. bassiana IMI 382302, although however, weak activity was detected against T. harzianum T24 only at the low conidial concentration but not at the higher one (1 × 10⁸ ml^?1). Thus, this study concludes that larvae of S. littoralis showed immune-dependant sensitivity to T. harzianum T24 and B. bassiana IMI 382302. Therefore, this study may recommend these two fungal isolates as mycoinsecticides in the battle against cotton leaf worm in Egypt. Hence, they have been selected for future comprehensive bioassays in the laboratory under conditions similar to that in the field. This, in fact, may help for developing effective mycoinsecticides against this pest. Penetration mechanims of insect cuticle by entomopathogenic fungi will be discussed.     

Selection of brown spot-resistant rice plants from Helminthosporium oryzae toxin-resistant calluses

Helminthosporium oryzae toxin induced electrolyte leakage from rice callus tissues and caused their browning and death. A virulent isolate of the pathogen invaded and colonised callus tissues rapidly, while a less virulent and a nonpathogenic isolate colonised calluses only weakly if at all. Addition of the toxin to calluses permitted colonisation of calluses by the nonpathogenic isolate. Four toxin-resistant calluses were selected and plants regenerated from two of these resistant calluses showed resistance to the pathogen. This resistance was heritable and stability of resistance was observed in the R₁, R₂ and R₃ generations.     

Morphological and physiological comparisons ofHelicobasidium mompa andH. purpureum

Morphological and physiological comparisons were made of sevenHelicobasidium mompa isolates and fourH. purpureum isolates. Colonies of theH. mompa isolates were thin, dense, or hard and dense, and most were pale brown to brown or dark brown, while that of isolate 344c was pinkish. Colonies ofH. purpureum isolates were hard and dense, and their colonies were dark brown. Diameters of hyphae were similar forH. mompa andH. purpureum. Dimensions of conidia and morphology of conidiophores ofH. mompa isolate 344c were close to those ofH. purpureum reported previously.H. mompa isolates grew well at 23°C, 25°C or 27°C, while all isolates ofH. purpureum grew well at 23°C. Growth rates ofH. purpureum isolates was almost the same as those ofH. mompa isolates with slow growth. Polygaracturonase activity at pH 3 was variable among the isolates for bothH. mompa andH. purpureum. Itaconic acid was produced abundantly by three isolates ofH. mompa but not produced by isolate AH130, whereas all isoaltes ofH. purpureum produced a small amount of itaconic acid.     

LABORATORY SUSCEPTIBILITY OF PLUTELLA XYLOSTELLA TO METARHIZIUM ANISOPLIAE AND NOMURAEA RILEYI*

Abstract Five isolates of Metarhizium anisopliae and one isolate of Nomuraea rileyi were bioassayed against larvae of Plutella xylostella. Larvae were treated by exposing cabbage leaf discs previously immersed in conidial suspension, and were then incubated at 25^oC and observed daily. One of M. anisopliae isolates, F11248, originally from Mastotermes darwiniensis was found being most virulent against P. xylostella. The LC₅₀ was estimated as 2.03 times 10⁴conidia/ml with 9 d mortality data, and the LT₅₀ was 4.97 d at concentration of 10⁷conidia/ml. Isolate F163 (N. rileyi) showed virulence to P. xylostella in both tests, but no cadavers sporulated.     

A new technique for monoconidial culture of the most aggressive isolate in a given population of Bipolaris sorokiniana, cause of foliar spot blotch in wheat and barley

We developed a new technique for monoconidial culture of the most aggressive isolate in a given population of Bipolaris sorokiniana, to facilitate the evaluation of spot blotch resistance in wheat and barley. Blotched portions of infected barley leaves were placed on a glass slide in a moist chamber for production of conidia by associated fungal hyphae. Conidia were collected separately and grown on water agar discs. Individual water agar discs having conidium growth were inoculated on barley leaves. The conidium producing the earliest symptom with the largest lesion was considered most aggressive. This lesion was incubated in a moist chamber and the conidial offspring were tested for pathogenicity. When a uniform infection was observed, a small piece of the lesion was cut using a sterilized scalpel, surface sterilized with NaOCl, and inoculated in the centre of Petri dishes containing potato dextrose agar medium. The inoculated Petri dishes were incubated at 25 ± 1 °C to yield monoconidial cultures of the most aggressive isolate. Variability in symptom expression caused by the most aggressive isolate of a given population was much less than variability in symptom expression caused by all isolates collectively. The techniques will be useful for plant pathologists and breeders in screening for spot blotch resistance in wheat and barley.     

Allee Effect in the Infection Dynamics of the Entomopathogenic Fungus Beauveria bassiana (Bals) Vuill. on the Beetle, Mylabris pustulata

Successful infection by Beauveria bassiana as with all other entomopathogenic fungi, is accomplished only at a high conidial dose while, theoretically, a single conidium should be sufficient. Indeed, this is a major deterrent in its use as a biocontrol agent. High pathogen load for infection is required by organisms which display ‘Allee’ effect. In such organisms, a threshold exists for pathogen dose, below which no infection can be caused. B. bassiana has a semelparous life cycle and, therefore, its infection dynamics are expected to conform to the mass action principle with a linear relationship between dose and successful infection observable as mortality of the insect. Whether the need for a high conidial dose to induce insect mortality by B. bassiana is due to the operation of Allee effect was examined. A sample of 34 isolates was bioassayed on Mylabris pustulata (Coleoptera: Meloidae) at four conidial concentrations. With more than half of the isolates in the sample, the lowest dose tested (10⁴ conidia/insect) did not cause insect mortality. Thus, a threshold pathogen load is required to cause successful infection. In these isolates, the dose–mortality relationship was sigmoid. Allee effect is thus identified in the infection dynamics of B. bassiana–M. pustulata system. The isolates that induced mortality at the lowest dose tested are concluded to be highly virulent with a lower threshold dose required for successful infection. With some isolates, at high conidial dose, the infection rate decreased either due to a decrease in the proportion of insects showing mycosis, to the speed of death, or both. Such a response could result from intra scramble competition arising from overload of pathogen at very high dose.     

Combining vegetable oil and sub-lethal concentrations of Imidacloprid with Beauveria bassiana and Metarhizium anisopliae against adult guava weevil Conotrachelus psidii (Coleoptera: Curculionidae)

Of the insect pests that attack guava fruits, the guava weevil, Conotrachelus psidii Marshall, 1922 (Coleoptera: Curculionidae), is one of the most important in Brazil. In search of alternatives to chemical pesticides, this study was performed to select fungal isolates of Beauveria bassiana and Metarhizium anisopliae as potential candidates for the control of adult C. psidii. Tests were carried out using three products applied with the entomopathogenic fungi: Tween 80, sunflower oil and Imidacloprid (IMI). A sub-lethal concentration of IMI was determined (100 ppm). The results demonstrated that LPP 19 and LPP 114 were the most effective isolates when used in combination with all of the products. The least virulent isolate, ESALQ 818, when applied in Tween 80 caused only 26.6% mortality, however, this isolate showed significantly improved efficiency when applied together with either sunflower oil or IMI, causing 57.3 and 88.6% mortality, respectively. The efficiency of all the isolates tested here improved when applied together with IMI, with LT₅₀ values of 5.3–10.3 days when compared to LT₅₀ values in Tween alone of 9.5–17 days. The isolate that produced the highest number of conidia on the cadavers of adult C. psidii was LPP138, independent of the product used; however, conidial production was slightly reduced when fungi were applied together with IMI. These results are promising for developing new formulations of the isolates to be tested in the field.     

Laboratory evaluation of entomopathogenic fungi against the white grubs,Holotrichia oblita and Anomala corpulenta (Coleoptera: Scarabaeidae) from the field of peanut,Arachis hypogaea

We evaluated the pathogenicity of nine isolates of entomopathogenic fungi, including six isolates of Metarhizium anisopliae, one of Beauveria bassiana, and two of Beauveria brongniartii, against eggs and various larval instars of two scarab-beetle species, Holotrichia oblita and Anomala corpulenta, under laboratory conditions. The fungal isolates differed in pathogenicity. Generally, the isolates were more pathogenic to A. corpulenta than to H. oblita. Some of the isolates prevented egg hatching and caused larval death. Isolates M2-2 and Br5-8 caused 39 and 27% egg mortality, respectively, and produced 23 and 24% viewable fungal-infection rates in H. oblita. Three isolates had no significant effect on egg hatchability. Three isolates (Br5-8, Br232818, and M200614) caused about 40% mortality in H. oblita first instars. In A. corpulenta, all isolates except M200614 caused more than 60% egg mortality, and M2-2, Br232818 and Br5-8 caused egg-infection rates greater than 50%. M2-2 caused 47% infection and 100% mortality in first-instar larvae of A. corpulenta, while Br5-8 and Br232818 yielded over 80% mortality of the larvae. The three most virulent isolates, M2-2, Br232818 and Br5-8, were selected for further bioassays against second- and third-instar larvae. In addition, seven graduated concentrations of a Br5-8 conidial suspension were assayed against H. oblita second instars. Larval mortality was positively correlated with fungal dosage, and the LC₅₀ was 4.49×10⁶ conidia/mL. These three virulent isolates may be used to prevent H. oblita and A. corpulenta larval infestations in field crops.