Entomopathogenic fungi-mediated biological control of the red palm weevil Rhynchophorus ferrugineus

The red palm weevil (RPW), Rhynchophorus ferrugineus, is an important pest of palms, and difficult to control by conventional methods. Therefore, microbial control is an alternative strategy for controlling RPW. Herein, a total of 15 entomopathogenic fungi (EPFs) were subjected to primary pathogenicity screening against last stage of RPW larvae. The preliminary data showed that four Beauveria bassiana isolates (JEF-484, 158, 462 and 507) and one Isaria fumosorosea isolate (JEF-014) resulted in 100 % mortality within 5–10 days post inoculation (d.p.i.), respectively. According to the time required for RPW mortality, JEF-484, 158, 462 and 014 were further subjected to bioassays using 10⁷ conidia/ml suspensions by spraying method. Based on the results, JEF-484 showed the highest mortality and shortest LT₅₀ on the last stage of RPW larvae, followed by JEF-158. The two isolates also showed good conidial production and high thermal stability compared to the other isolates. Therefore, JEF-484 and JEF-158 were selected for bioassays against RPW egg and the last larval stage with different concentrations of 10⁵, 10⁶ and 10⁷ conidia/ml conidial suspensions by spraying method. For the bioassay at the egg stage, JEF-158 showed a significantly higher ovicidal effect than JEF-484. In the larval bioassay, both EPF isolates showed a dosage-dependent effect on the RPW larvae. JEF-484 caused higher mortality in RPW larvae than JEF-158. In summary, the combination of the 2 promising EPF isolates might provide an opportunity for the practical microbial control of RPW at different life stages in palm tree fields.     

Pathogenicity, formulation and storage of insect pathogenic hyphomycetous fungi tested against Diabrotica speciosa

Studies were conducted tosearch for fungal strains with potentialpathogenicity against Diabrotica speciosa(Germar) (Coleoptera: Chrysomelidae).Among sixteen fungal isolates screenedthe most virulent was a Beauveria bassiana(Balsamo) Vuillemin isolate (FHD13) thatcaused 70% mortality of D. speciosathird instar larvae. The LC₅₀ value ofB. bassiana isolate FHD13 was3.48 × 10¹⁰ conidia/ml.Different temperatures (4, 17 and 26 °C)and vegetable oils (corn, sunflower and canola)used for storage did not significantly affectviability of conidia. A pathogenicity trialagainst D. speciosa larvae performed withthe corn oil formulation (1 × 10⁸ conidia/mlof oil) caused 65% of mortality.     

Susceptibility of Larvae of Galleria mellonella to Infection by Aspergillus fumigatus is Dependent upon Stage of Conidial Germination

The ability of conidia of the human pathogenic fungus Aspergillus fumigatus to kill larvae of the insect Galleria mellonella was investigated. Conidia at different stages of the germination process displayed variations in their virulence as measured using the Galleria infection model. Non-germinating (‘resting’) conidia were avirulent except when an inoculation density of 1 × 10⁷ conidia per insect was used. Conidia that had been induced to commence the germination process by pre-culturing in growth medium for 3 h were capable of killing larvae at densities of 1 × 10⁶ and 1 × 10⁷ per insect. An inoculation density of 1 × 10⁵ conidia per insect remained avirulent. Conidia in the outgrowth phase of germination (characterised as the formation of a germ tube) were the most virulent and were capable of killing 100% of larvae after 5 or 24 h when 1 × 10⁷ or 1 × 10⁶ conidia, that had been allowed to germinate for 24 h, were used. Examination of the response of insect haemocytes to conidia at different stages of the germination process established that haemocytes could engulf non-germinating conidia and those in the early stages of the germination process but that conidia, which had reached the outgrowth stages of germination were not phagocytosed. The results presented here indicate that haemocytes of G. mellonella are capable of phagocytosing A. fumigatus conidia less than 3.0 μm in diameter but that conidia greater than this are too large to be engulfed. The virulence of A. fumigatus in G. mellonella larvae can be ascertained within 60–90 h if infection densities of 1 × 10⁶ or 1 × 10⁷ activated conidia (pre-incubated for 2–3 h) per insect are employed.     

Lethal effects of Gliocladium virens,Beauveria bassiana and Metarhizium anisopliae on the malaria vector Anopheles albimanus (Diptera: Culicidae)

Control of Anopheles albimanus, the main vector of malaria on the coast of the State of Chiapas, is based mainly on application of chemical insecticides, which has resulted in resistance to most registered insecticides. Strategies for biological control may provide sustainable alternatives. We report on the lethal effects of a native isolate of Gliocladium virens on An. albimanus larvae and adults, compared to that of strains of Beauveria bassiana and Metarhizium anisopliae. Conidial suspensions of G. virens, B. bassiana and M. anisopliae cultured on Sabouraud agar were tested in bioassays with An. albimanus larvae and adults. Mosquito larvae were more susceptible to all fungi, compared to adults. On early and late instar larvae, M. anisopliae showed the most pathogenic effect (LC₅₀ of 1.4×10⁵ conidia/mL in early instars; 1.1×10⁵ conidia/mL in late instars), followed by G. virens (LC₅₀ of 3.3×10⁵ conidia/mL in early instars and 3.5×10⁶ conidia/mL in late instars). Metarhizium anisopliae sensu lato and the native G. virens could be considered good choices for An. albimanus control in southern Mexico.     

Isolation and characterization of a Serratia marcescens with insecticidal activity from Polyphylla olivieri (Col.: Scarabaeidae)

Members of the genus Serratia are known for their abilities to infect insects. In this study, a red‐pigmented S. marcescens was isolated and characterized from the infected larvae of Polyphylla olivieri using bacterial cultivation, phylogenetic analysis as well as bioassays against larvae of the two insect pests, Plodia interpunctella and Ephestia kuehniella. Comparative 16S rRNA and groEL gene sequence BLAST analyses strongly suggested that the isolated strain should be placed in the genus Serratia, sharing high sequence similarities with several strain of S. marcescens associated with insects. Phylogenetic analysis placed the isolated bacterium with other S. marcescens bacteria in a clade with high bootstrapping values. To assess pathogenicity of the S. marcescens isolate, the bacterial cells were either injected into the haemolymph of the fifth‐instar larvae or added to the diets of insects. Survival curves of the control insects and those challenged with six different concentrations of S. marcescens showed that the S. marcescens isolate significantly reduced survival rates of the larvae. The LC₅₀s of the bacterium on P. interpunctella and E. kuehniella were 1992.26 and 1.09 × 10⁴ (CFU/μl) for injection bioassays at 6 h post‐injection, and 4.48 × 10⁴ and 1.96 × 10⁵ (CFU/10 μl) for feeding bioassays at 24 h post‐feeding, respectively. Injection of the bacterial culture supernatant into the larvae led to continuous bleeding from the site of injection, while injection of heat‐treated culture supernatant of the bacterium did not cause continuous bleeding. Together, our results showed the possibility of using this S. marcescens isolate in microbial control of the insect pests after addressing the safety concerns. Moreover, it might be considered as a source of useful bioactive molecules and genes with application in insect control and biotechnology via developing insect‐resistant plants.     

Nucleopolyhedrovirus infection and/or parasitism by Microplitis pallidipes affected haemolymph titre of 20‐hydroxyecdysone in Spodoptera exigua larvae

This study examined the physiological effects of joint and separate parasitism and infection by the endoparasitoid Microplitis pallidipes Szépligeti and the nucleopolyhedrovirus (NPV), respectively, on haemolymph 20‐hydroxyecdysone (20‐E) titre in Spodoptera exigua (Hübner) larvae. The results indicated that in parasitized larvae, virus‐infected larvae (5.7 × 10³ and 5.7 × 10⁵ OB/ml) and parasitized larvae infected with virus at 5.7 × 10⁵ OB/ml, compared to healthy larvae, the 20‐E all declined during the first 3 days but began to increase from day 4 after treatment, while in jointly parasitized and infected larvae (5.7 × 10³ OB/ml), the 20‐E declined during the first 4 days but began to increase on day 5 after treatment. Meanwhile, compared to parasitized larvae, the 20‐E declined during the first 4 days but significantly increased on day 5 in jointly parasitized and infected larvae (5.7 × 10³ OB/ml), while significantly increased during the first 2 days but began to decrease from day 3 after treatment in jointly parasitized and infected larvae (5.7 × 10⁵ OB/ml). Finally, in larvae that were both parasitized and virus infected (5.7 × 10³ OB/ml), compared to just virus‐infected larvae (5.7 × 10³ OB/ml), the 20‐E was lower on days 3 and 4 but higher on other days after treatment; in larvae that were both parasitized and virus infected (5.7 × 10⁵ OB/ml), compared to just virus‐infected larvae (5.7 × 10⁵ OB/ml), the 20‐E was significantly higher at the first 2 days but lower from day 3 after treatment. Our results revealed that 2nd instar larval M. pallidipes in host bodies may release 20‐E into the haemolymph of S. exigua larvae and that NPV infection may stimulate S. exigua to release more 20‐E during its third to fourth instar larval moulting. We found that this stimulatory effect was greater with higher virus concentrations.     

Pathogenicity of entomopathogenic fungus,Metarhizium anisopliae MET-GRA4 isolate on dengue vectors,Aedes albopictus and Aedes aegypti mosquito larvae (Diptera: Culicidae)

Considering the rapid transmission of the dengue virus, substantial efforts need to be conducted to ward-off the epidemics of dengue viruses. The control effort is depending on chemical insecticides and had aroused undesirable conflicts of insecticide resistance. Here, we study the entomopathogenic fungus, Metarhizium anisopliae as a promising new biological control agent for vector control. The pathogenicity effects of Metarhizium anisopliae against field and laboratory strains of Aedes albopictus and Aedes aegypti larvae were tested using the larvicidal bioassay technique. The results demonstrate that the treatments using M. anisopliae isolate MET-GRA4 were highly effective and able to kill 100% of both Ae. albopictus and Ae. aegypti mosquito larvae at a conidia concentration of 1 × 10?/ml within 7 days of the treatment period. The fungus displayed high larvicidal activity against laboratory and field strain of Ae. aegypti larvae with LC₅₀ values (9.6 × 10³/ml, 1.3 × 10³/ml) and LC₉₅ values (1.2 × 10?/ml, 5.5 × 10⁵/ml) respectively. For Ae. albopictus, LC₅₀ values for laboratory and field strains were (1.7 × 10⁴/ml, 2.7 × 10⁴/ml) and the LC₉₅ values were (2.1 × 10?/ml, 7.0 × 10⁵/ml) respectively. Interestingly, the susceptibility of field strain towards M. anisopliae was higher as compared to the laboratory strain Aedes larvae. In which, the causative agents of all the dead larvae were verified by the virulence of M. anisopliae and caused morphological deformities on larval body. The findings from this study identify this isolate could be an effective potential biocontrol agent for vector mosquitoes in Malaysia.     

Time-concentration-mortality response of potato tuber moth pupae to the biocontrol agent Cordyceps tenuipes

ABSTRACT

The potato tuber moth (PTM) Phthorimaea operculella is a critical potato pest. Larvae infest both foliage and tubers and mature larvae pupate in the soil or other safe places. Cordyceps tenuipes, an entomopathogenic fungus, infect lepidopteran pupae. To determine the effectiveness of this fungus as a biocontrol agent for PTM, we evaluated the time-concentration-mortality (TCM) response of PTM pupae to C. tenuipes using the following bioassays: (1) direct immersion in conidial suspensions, (2) incubation in sterilised or (3) unsterilised soilpremixed with conidia, and (4) incubation in unsterilised soil drenched with conidial suspensions to simulate field conditions. Fungal infection caused 100%, 83.3%, 73.3%, and 85.0% mortality of PTM pupae in assays 1–4, respectively. At 10⁸ conidia/mL or conidia/g concentration, assays 1 and 4 had short lethal times (LT₅₀) of 2.2 and 2.6 days compared with 3.7 and 4.8 days for assays 2 and 3, respectively. On day 7 after inoculation, assays 1 and 4 also had low lethal concentrations (LC₅₀) of 1.69 × 10³ conidia/mL and 1.10 × 10⁵ conidia/g compared with those of assays 2 and 3, which showed low virulence, with LC₅₀ of 3.50 × 10⁵ and 3.60 × 10⁶ conidia/mL, respectively. Our results demonstrate that C. tenuipes is a promising candidate for PTM biocontrol at the pupal stage. Drenching the soil surface with conidial suspensions may be the most effective method of field application.     

Virulence of a South African isolate of the <Emphasis Type="Italic">Cryptophlebia leucotreta</Emphasis> granulovirus to <Emphasis Type="Italic">Thaumatotibia leucotreta</Emphasis> neonate larvae

Surface inoculation dose–response and time–response bioassays and detached fruit bioassays were conducted with a novel South African isolate of the Cryptophlebia leucotreta granulovirus (CrleGV-SA) against Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Noctuidae) neonate larvae. LC₅₀ and LC₉₀ values were estimated to be 4.095 × 10³ and 1.185 × 10⁵ OBs ml⁻¹, respectively. LT₅₀ and LT₉₀ values were estimated to be 4 days 22 h and 7 days 8 h, respectively, categorising the virus as a fast or type 2 granulovirus. There was a conspicuous difference in behaviour between larvae on inoculated diet and untreated diet, resulting in a significant reduction in penetration of diet. Bioassays on detached Navel oranges revealed LC₅₀ and LC₉₀ values of 9.310 × 10⁷ and 1.515 × 10⁹ OBs ml⁻¹, when using data on numbers of larvae per fruit rather than on numbers of infested fruit. Field trials will be conducted.     

Molecular and Morphological characterization of multiple nucleopolyhedrovirus from Mexico and their insecticidal activity against Spodoptera frugiperda (Lepidoptera: Noctuidae)

The fall armyworm (FAW), Spodoptera frugiperda (Lepidoptera: Noctuidae), is the most important pest of maize in many countries. Entomopathogenic viruses mainly Baculoviruses family are excellent biological control agents and therefore a viable alternative for managing this pest. The aim of this study was to determine the biological activity of eight native nucleopolyhedrovirus (NPVs) against FAW larvae. Additionally, two of the most virulent isolates (SfCH32 and SfCH15) were characterized biologically by bioassays to estimate their median lethal dose (LD₅₀) and median lethal time (LT₅₀), morphologically by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) and molecularly by restriction enzymes. Three (SfCH15, SfCH18 and SfCH32) of the eight tested native nucleopolyhedrovirus isolates caused mortalities ˃98% at 168-hr post-inoculation (hpi) with a dose of 9.2 × 10⁴ OBs/larva. SfCH15 and SfCH32 isolates showed occlusion bodies (OBs) of irregular shape and size (1.02–2.24 μm). The SfCH15 and SfCH32 isolates showed similar median lethal dose (5.6 × 10²–6.4 × 10² OBs/larva). The lowest median lethal time (114.5 hpi) was observed with the SfCH15 isolate at the highest concentration (2.5 × 10⁶ OBs/larva). The DNA restriction profiles for SfCH15 and SfCH32 were different, with their genome size being ~128,000 bp and 132,000 bp, respectively. SfCH15 and SfCH32 isolates showed similar morphological characteristics and the highest virulence against fall armyworm. This study showed that native isolates were highly virulent against S. frugiperda larvae, being similar to other reported strains; however, field studies are required to confirm their insecticidal effect.     

Two strains of Pseudomonas fluorescens bacteria differentially affect survivorship of waxworm (Galleria mellonella) larvae exposed to an arthropod fungal pathogen,Beauveria bassiana

Two strains of Pseudomonas fluorescens were found contaminating a biopesticide used in a previous study against Varroa destructor infestations in honey bee hives. In that study, the biopesticide, a formulation of a fungal pathogen of arthropods, Beauveria bassiana, failed to have any negative impact on the mite infestation despite successful results in previous studies using uncontaminated batches of the same biopesticide. The objective of the present research was to determine whether the bacteria may have interfered with the infectivity and/or virulence of B. bassiana in a simplified system; positive results in that system would then provide a rationale for further work under more complex conditions. Galleria mellonella late instar larvae treated topically with both a bacterial suspension of 6.8 to 7.0×10⁷ cfu/ml and a fungal suspension of 2.5×10⁷ or 2.5×10⁸ B. bassiana conidia/ml showed, in the case of one of the bacterial strains, significantly increased survivorship compared to larvae treated with just the B. bassiana suspension. When larvae were immersed in a bacterial suspension prior to application of B. bassiana suspension using a spray tower, a significant positive effect of the same P. fluorescens strain on larval survivorship was observed at 2.5×10⁸ conidia/ml. Neither the bacterial suspensions alone nor blank control solutions had any effect on larval survivorship. These results show that an interaction between the bacteria and the pathogen may explain some of the results from the prior field trial.     

Efficacy of Beauveria bassiana (Bals.) Vuill. against the tarnished plant bug, Lygus lineolaris L., in strawberries

Beauveria bassiana has a high insecticidal potential to control the tarnished plant bug, Lygus lineolaris, a significant pest of strawberries. Screening experiments showed that L. lineolaris adults were susceptible to several B. bassiana isolates. Another screening test with Coleomegilla maculata, a natural enemy found in strawberries, was also performed in order to select the isolate having lower entomopathogenic impact on this insect. Based on data obtained from both insect species and on the ecozone origin of the B. bassiana isolates, INRS‐IP and INRS‐CFL isolates were selected for further experiments. The LC₅₀ values of these two isolates against L. lineolaris adults were 7.8 × 10⁵ and 5.3 × 10⁵ conidia/ml, and average survival time (AST) values were 4.46 and 4.37 days at a concentration of 1 × 10⁸ conidia/ml respectively. Results also indicated that L. lineolaris nymphs are susceptible to the selected isolates. During field experiments, using a randomized block design with four replicates, INRS‐IP and INRS‐CFL isolates were applied at two rates (1 × 10¹¹ and 1 × 10¹³ conidia/ha) weekly during a period of 4 weeks. These multiple applications triggered a significant reduction of L. lineolaris nymphal populations in strawberries. Twenty‐four days after the first application, a significant difference was observed between the mean population densities of surviving nymphs in all B. bassiana‐treated plots (less than one insect per five plants) compared with those in control plots (four insects per five plants). During the field experiment, persistence of insecticidal activity and viability of B. bassiana conidia were also monitored. The results showed the presence of viable and infective conidia up to 6 days after each application on strawberry foliage. Moreover, the multiple applications of B. bassiana at the rate of 1 × 10¹³ conidia/ha triggered a significant reduction in strawberry fruit injuries induced by L. lineolaris feeding behaviour compared with the control plots.     

A new microbial consortia containing entomopathogenic fungus,Beauveria bassiana and plant growth promoting rhizobacteria,Pseudomonas fluorescens for simultaneous management of leafminers and collar rot disease in groundnut

The virulence of 20 isolates of Beauveria bassiana (Balsamo) Vuillemin to larvae of the leafminer, Aproaerema modicella, was tested in the laboratory. Leafminer larvae were sprayed with a standard concentration of 1×10⁸ condia/mL of each B. bassiana isolate. All the B. bassiana isolates tested were pathogenic to A. modicella and the mortality varied between 16.7 and 68.9%. Beauveria bassiana isolate B2 was found to be the most virulent followed by isolate B4 which resulted in 59% mortality. Beauveria isolate B2 was selected for dose–response mortality studies with four different doses (1×10², 1×10⁴, 1×10⁶ and 1×10⁸ conidia/mL). Among the various doses tested, 1×10⁸ conidia/mL produced the highest mortality (70.7%). In addition, morphogenesis of the insect pest in all stages, larval, pupal and adult was greatly affected due to fungal infection. Further, B. bassiana isolate B2 and two Pseudomonas fluorescens strains, TDK1 and Pf1 were tested alone and in combination for suppression of groundnut leafminer and collar rot disease and promotion of plant growth and yield both under glasshouse and field conditions. The mixture of B. bassiana and P. fluorescens strains significantly reduced the leafminer and collar rot disease incidences when applied as talc-based formulation through seed, soil and foliar application under glasshouse and field conditions.     

The reproduction potentials of four entomopathogenic nematode strains related to cost-effective production for biological control

Bioassays to evaluate the mortality, virulence and reproduction potentials of four indigenous EPN strains, S-PQ16, S-BM12, H-KT3987 and H-CB3452 on insect larvae of mealworm (Tenebrio molitor) and greater wax moth (Galleria mellonella) revealed the highest mortality rates of two insect larvae at the highest inoculation dose of 100 IJs to range from 89 to 100 percent and 94.3–100 percent at 48 h after inoculation, respectively. Virulence was high for all nematode strains, with LC₅₀ values between 29.6 and 47.3 IJs/insect host. The highest IJ yields were different between nematode strains and insect host, from 66.8 × 10³ IJs (S-PQ16) to 118.6 × 10³ IJs (H-KT3987) on T. molitor, and from 54.2 × 10³ IJs (S-BM12) to 163.3 × 10³ IJs (H-KT3987) on G. mellonella. The culturing cost in terms of food expenditure for rearing insect larvae varied between insect larvae and nematode strains, from 6.76 to 26.63 USD per billion IJs for nematode strains cultured on T. molitor larvae and from 3.54 to 7.81 USD per billion IJs for nematode strains cultured on G. mellonella larvae. The full cost for a nematode product of 2.5 × 10⁹ IJs per hectare, produced through in vivo mass culturing, of the most efficient nematode strain, H-KT3987, was 191.3 USD, slightly cheaper than 199.4 USD for the same nematode product produced through in vitro mass culturing.     

Cosmopolites sordidus (Germar) susceptibility to indigenous Cameroonian Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metsch.) isolates

Management of the banana root borer (BRB), Cosmopolites sordidus (Germar; Coleoptera: Curculionidae), remains a challenge in banana and plantain production worldwide. Synthetic pesticides remain the most widely used solution while mycoinsecticides are increasingly being recommended. In this study, we selected indigenous isolates of Beauveria bassiana and Metarhizium anisopliae collected from plantain fields in Cameroon, and tested them in the laboratory for their viability, pathogenicity and virulence against all C. sordidus life stages. Of 13 isolates initially screened for spore germination and pathogenicity to adult weevils in conidial suspension of 3.2 × 10⁸ conidia/ml, eight isolates with high to moderate germination and highest weevil mortality were selected for dose–response bioassays with four concentrations per isolate: 3.2 × 10², 3.2 × 10⁴, 3.2 × 10⁶ and 3.2 × 10⁸ conidia/ml. The virulent isolates from adult bioassays were tested with eggs, larva and pupae in conidial suspension of 3.2 × 10⁸ conidia/ml. Isolates performance depended on insect life stage with significantly high pathogenicity and virulence against larval, pupa and adult stages. The Beauveria isolate BIITAC6.2.2 caused the highest mortality rates followed by MIITAC1.1.5. Lethal times and lethal concentrations were relatively low for the three M. anisopliae isolates and three B. bassiana isolates which were the best isolates in almost all insect life stages. Apart from being effective in multiple life stages, these isolates were transmitted horizontally from one stage to another when eggs and pupae were treated. The implication of these findings for integrated management of the BRB, and potential biopesticides development and commercialization are discussed.     

Combination of the fungus Beauveria bassiana and pheromone in an attract‐and‐kill strategy against the banana weevil,Cosmopolites sordidus

An attract‐and‐kill approach based on pellets from soybean or palm stearin fats blended with the entomopathogenic fungus Beauveria bassiana (Bals.) Vuill. sensu lato and the aggregation pheromone sordidin (Cosmolure^®) was tested against the banana weevil, Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae). The viability of B. bassiana conidia, blended with hydrogenated oil and exposed for up to 150 min to heating at 50 °C, was not affected and the aggregation pheromone did not undergo any decomposition. Conidial viability in pellets decreased by 50% after an average of 15.1 and 9.1 days at 25 and 40 °C, respectively, when packaged in polypropylene bags. Active packaging (hermetic bag + O₂/moisture‐absorbing sachet) increased the shelf lives almost 10 and 6 times at 25 and 40 °C, respectively. In olfactometer bioassays, fat pellets amended with pheromone (sordidin, 1% wt/vol) were highly attractive to C. sordidus adults for up to 15 days, after which the pheromone release rate had decreased by about 90% and pellets were no longer attractive. Pellets with pheromone and conidia were as attractive to C. sordidus as banana rhizomes, and considerably more attractive than pieces of pseudostem. In no‐choice experiments conducted in boxes, survival of insects exposed to fungus‐impregnated pellets was affected by fat type (soybean fat vs. palm stearin) and bioassay temperature (25 vs. 30 °C), with results favoring soybean fat pellets at the higher temperature (96.9% of mortality after 18 days and ST₅₀ of 7.7 days). However, mortality levels were low (21.7% for soybean fat pellets) or very low (1–5% for palm stearin pellets) in choice experiments carried out at 25 °C when fungus‐impregnated pellets were applied before or after exposure of pseudostem residues to insects, respectively. The potential of this delivery system to manage C. sordidus populations and other insect pests (including those with cryptic habits) is discussed.     

Coconut malformation: An emerging disease caused by Fusarium proliferatum in southern Iran

Symptoms of vegetative malformation were observed on coconut palms (Cocos nucifera L.) in the Qeshm Island, Bandar Abbas and Minab, in Hormozgan province, southern Iran. The symptoms included misshapen and dwarfed leaves with shortened, thickened and tightened leaflets in wavy and zigzag form. The aim of this study was to identify the causal pathogen of coconut palm malformation and complete Koch's postulates for putative pathogen. Small pieces of surface‐disinfested malformed vegetative tissues of coconut palms were cultured on potato dextrose agar (PDA) medium. Fusarium isolates were permanently obtained from the symptomatic tissues. Sequence data from the internal transcribed spacer region (ITS1–5.8S‐ITS2) and translation elongation factor 1 alpha (TEF‐1α) gene were used for molecular identification of the isolates. BLAST search of the sequences showed 99%–100% identity to several Fusarium proliferatum strains in the GenBank, FUSARIUM‐ID and Fusarium MLST databases. A phylogeny inferred using individual sequence data from ITS region and TEF‐1α gene placed our isolates together with the other F. proliferatum sequences retrieved from the GenBank. Pathogenicity tests were carried out using one‐year‐old healthy coconut palm seedlings and conidial suspensions (10⁶ conidia/ml) of the F. proliferatum isolates. The first visible symptoms appeared on newly produced leaves of the inoculated seedlings during the 16th week after inoculation, wherease no disease symptoms were observed on the control plants until the end of the experiment. Reisolation from symptomatic tissues of the inoculated seedlings yielded isolates of F. proliferatum with morphological and molecular characteristics identical to those of the isolates used for inoculations. This is the first report of coconut palm malformation caused by F. proliferatum worldwide.     

Variability in virulence of Beauveria spp. soil isolates against Ostrinia nubilalis

European corn borer (Ostrinia nubilalis) is an important maize pest which is treated mainly by synthetic pesticides with many negative effects for ecosystem and human health. Soil borne entomopathogenic fungi of species belonging to Beauveria genus may provide an alternative strategy for biological control of this pest. In this article, we report results of virulence testing of 46 soil isolates of Beauveria spp. against O. nubilalis in relation to isolates origin and haplotype. All strains were collected from soil using Galleria mellonella bait method. Conidia suspension with concentration 10⁷ conidia in ml was used for virulence test against 4th instar larvae of O. nubilalis at temperature 25°C. After 14 days, mortality of larvae was in the range 34%–96%. Median lethal time LT50 was estimated to 5.5–21.3 days. Mortality was not in relation to habitat or any other environmental parameter of sites where isolates came from. Differences among species were insignificant. Isolates were divided into 8 genetic lineages and 14 haplotypes using sequencing of ITS and Bloc nuclear regions. None of the lineages showed higher mortality than others. Despite more or less virulent isolates being found in each lineage, the three most virulent isolates belonged to the same lineage Beauveria bassiana A3. The four most virulent isolates were compared to commercial isolate GHA to assess medium lethal concentration. LC50 were estimated in range 7.5 × 10⁴ to 9.5 × 10⁵. Three of the four isolates showed equal or greater efficiency than strain GHA.     

Some studies on Nigerian palm wine with special reference to yeasts

The identification and enumeration of yeasts and the effect of chemical preservatives on the yeast load in Nigerian palm wine have been studied. Yeast found largely belong to the genus Saccharomyces. Other genera found were Candida, Endomycopsis, Hansenula, Kloeckera, Pichia, Saccharomycoides and Schizosaccharomyces. The viable yeast count ranged from 0.5 × 10⁷ cfu/ml to 4.2 × 10⁹ in both fresh Elaeis and Raphia spp. of palms. Yeasts in palm wine were least sensitive to sodium nitrate and most sensitive to sodium benzoate. The addition of these two chemical preservatives amongst others reduced the yeast count to 3.2 × 10⁸ (in Raphia sp.), and 4.1 × 10⁹ (in Elaeis sp.) with 0.025% sodium nitrate and 1.2 × 10⁶ (in Raphia sp.) and 1.9 × 10⁷ (in Elaeis sp.) with 0.1% sodium benzoate. The values of the yeast count in bottled and fresh palm wine were between 1.3 × 10³ cfu/ml to 9.8 × 10⁶ and 0.5 × 10⁷ to 4.2 × 10⁹, respectively. Maximum values of actual dry and theoretical yeast weights were 1.09 and 42 mg/ml, respectively.     

Pathogenicity of Beauveria bassiana and Metarhizium anisopliae to the tobacco spider mite Tetranychus evansi

Seventeen isolates of Metarhizium anisopliae (Metschnikoff) Sorokin and two isolates of Beauveria bassiana (Balsamo) Vuillemin were evaluated for their pathogenicity against the tobacco spider mite, Tetranychus evansi Baker & Pritchard. In the laboratory all the fungal isolates were pathogenic to the adult female mites, causing mortality between 22.1 and 82.6%. Isolates causing more than 70% mortality were subjected to dose–response mortality bioassays. The lethal concentration causing 50% mortality (LC₅₀) values ranged between 0.7×10⁷ and 2.5×10⁷ conidia ml⁻¹. The lethal time to 50% mortality (LT₅₀) values of the most active isolates of B. bassiana and M. anisopliae strains varied between 4.6 and 5.8 days. Potted tomato plants were artificially infested with T. evansi and treated with B. bassiana isolate GPK and M. anisopliae isolate ICIPE78. Both fungal isolates reduced the population density of mites as compared to untreated controls. However, conidia formulated in oil outperformed the ones formulated in water. This study demonstrates the prospects of pathogenic fungi for the management of T. evansi.