Fungal Pathogens Associated with Banana Fruit in Sri Lanka,and their Treatment with Essential Oils

The crown rot pathogens isolated from banana samples collected from 12 localities in Sri Lanka were Lasiodiplodia theobromae, Fusarium proliferatum and Colletotrichum musae. Fungal pathogens isolated were able to cause crown rot disease alone or in combination. Disease severity was higher when combinations of virulent pathogens were used. Cymbopogon nardus and Ocimum basilicum oils displayed fungicidal activity against C. musae and F. proliferatum between 0.2-0.6% (v/v) in a Poisoned food bioassay. Slightly lower concentrations of the test oils were needed for similar activity during liquid bioassays. The combination of Cymbopogon nardus and O. basilicum oils demonstrated synergistic action during both in-vivo bioassays.     

Effect of host quality of Callosobruchus maculatus (Coleoptera: Bruchidae) on performance of the egg parasitoid Uscana lariophaga (Hymenoptera: Trichogrammatidae)

Development and reproductive success of the solitary egg parasitoid Uscana lariophaga Steffan were examined after development in eggs of the bruchid storage pest Callosobruchus maculatus Fabricius reared at either low or high densities on cowpea seeds and laid at day 1 and 4 of maternal life. Both bruchid larval competition and maternal age negatively affected egg size, but the latter more than the former. Uscana lariophaga reared in small hosts developed slower, were smaller and produced fewer eggs compared to parasitoids reared in large hosts. Fecundity of the parasitoid was heavily influenced by host egg size. This was reflected in the values for the intrinsic rate of increase of U. lariophaga, which differed for wasps that developed in host eggs laid by bruchid females of different age. Wasps allocated marginally more female offspring to larger hosts.     

香茅精油对桔小实蝇产卵驱避作用及其化学成份分析

研究了香茅Cymbopogon nardus精油对桔小实蝇Bactrocera dorsalis (Hendel)的产卵驱避作用及其化学成分。结果表明,香茅精油对桔小实蝇产卵具有一定的驱避作用,不同浓度香茅精油处理芒果上的产卵量均显著低于对照,且随着香茅精油浓度的提高其产卵量逐渐减少,当浓度为10000μg/mL时,产卵驱避率为70.06%。采用气相色谱—质谱联用(GC-MS)技术分析了香茅精油的化学成份,结果发现该精油挥发物中含量较高的是3-蒈烯,香茅醛,香叶醇,香茅醇,柠檬烯。     

Characterization of resistance to Callosobruchus maculatus (Coleoptera: Bruchidae) in mungbean variety VC6089A and its resistance-associated protein VrD1

Characteristics of resistance of VC6089A, a mungbean, Vigna radiata (L.) Wilczek, bred by using a wild Vigna species, V. sublobata (Roxburgh) Verdcourt (accession no. TC1966), and containing a novel protein, VrD1, were investigated against the cowpea weevil, Callosobruchus maculatus (F.). The seeds of VC6089A showed high level of resistance; > 96% of the bruchid eggs failed to develop into adults, whereas 85% of eggs laid on susceptible cultivar VC1973A became adults. Mortality of surviving bruchids raised for five generations on VC6089A remained higher than 96%; however, female adults maintained high fecundity and thus showed a positive population growth through these generations. We therefore cannot exclude the possibility that the beetles could develop resistance to the resistant mungbean VC6089A. The protein VrD1 purified from seeds of VC6089A showed marked toxicity to C. maculatus when beetles were reared on artificial seeds containing varying levels of VrD1. Thorough inhibition of development was observed when artificial seeds containing 0.2% (wt:wt) VrD1 was provided for insect feeding. Our findings demonstrated the insecticidal activity of VC6089A mungbean seeds and VrD1 protein against C. maculatus. These results may facilitate safer control against bruchid infestation.     

Effects of Cymbopogon nardus (L.) W. Watson essential oil on the growth and morphogenesis of Aspergillus niger

The growth inhibitory effect of Cymbopogon nardus (L.) W. Watson var. nurdus essential oil on Aspergillus niger (Van Tieghem) mycelium was determined on agar medium. The mycelium growth was completely inhibited at 800 mg/L. This concentration was found to be lethal under the test conditions. Essential oil at 400 mg/L caused growth inhibition of 80% after 4 days of incubation, and a delay in conidiation of 4 days compared with the control. Microscopic observations were carried out to determine the ultrastructural modifications of A. niger hyphae after treatment with C. nardus essential oil. The main change observed by transmission electron microscopy concerned the hyphal diameter and the hyphal wall, which appeared markedly thinner. These modifications in cytological structure might be caused by the interference of the essential oil with the enzymes responsible for wall synthesis which disturb normal growth. Moreover, the essential oil caused plasma membrane disruption and mitochondrial structure disorganization. The findings thus indicate the possibility of exploiting Cymbopogon nardus essential oil as an effective inhibitor of biodegrading and storage-contaminating fungi.     

Bruchid (Coleoptera: Bruchidae) ovicidal phenylbutanoid from Zingiber purpureum

The larvicidal activity of the dichloromethane extract of Zingiber purpureum Roscoe (Zingiberaceae) rhizome against the second instar of Aedes aegypti (L.) (Diptera: Culicidae) is shown to be due to 4-(3',4'-dimethoxyphenyl)buta-1,3-diene. The diene also showed ovicidal activity against the bruchid Callosobruchus maculatus (F.) (Coleoptera: Bruchidae). Most of the eggs laid by bruchids on treated cowpea seeds were transparent, and very few of them contained developing embryos. The few larvae produced from these embryos were unable to penetrate the seed coat and enter the seed. Similar effects were seen when adults were exposed to the compound and then placed on untreated cowpea seeds, suggesting that a new type of maternally mediated ovicidal effect was involved. Coated and impregnated granular formulations of the extract were evaluated for use in the control of bruchid infestation of stored cowpea seeds. Coated granules showed activity similar to that of the crude extract but were found to lose activity rapidly. Impregnated granules were found to be less active than the crude extract.     

Mapping of quantitative trait loci for a new source of resistance to bruchids in the wild species Vigna nepalensis Tateishi & Maxted (Vigna subgenus Ceratotropis)

Azuki bean breeders have long been interested in producing azuki bean [Vigna angularis (Willd.) Ohwi & Ohashi] varieties with bruchid resistance. A new bruchid (Callosobruchus spp.) resistance source was found in V. nepalensis Tateishi & Maxted, a species that is cross compatible with azuki bean. Quantitative trait loci (QTLs) analysis for resistance to C. chinensis (L.) and C. maculatus (F.) was conducted using F(2) (V. nepalensis x V. angularis) and BC(1)F(1) [(V. nepalensis x V. angularis) x V. angularis] populations derived from crosses between the bruchid resistant species V. nepalensis and bruchid susceptible species V. angularis. Resistance was measured using two traits, percentage of seeds damaged by bruchids and the time taken for adult bruchids to emerge from seeds. Based on the results from both populations seven QTLs were detected for bruchid resistance; five QTLs for resistance to C. chinensis and two QTLs for resistance to C. maculatus. The different locations found for some resistance QTL to the two bruchid species suggests different resistance mechanisms. QTLs on linkage group (LG) 1 and LG2 for bruchid resistance to C. chinensis co-localized with seed size QTLs suggesting that incremental increase in seed size accompanied susceptibility to C. chinensis. Based on linked markers the QTL on these two linkage groups appear to be the same as previously reported in other Asian Vigna. However, several other QTLs were newly detected including one on LG4 that appears unrelated to seed size. Transfer of these new sources of bruchid resistance from V. nepalensis to azuki bean will be aided by the progress being made in azuki genome mapping.     

Insecticidal and biological efficacy of two vegetable oils against Callosobruchus maculatus (Fabricius) (Coleoptera: Bruchidae) under laboratory conditions

Bioefficacy of two vegetable oils, namely ethyl oleate and Acorus calamus, were conducted against the pulse beetle Callosobruchus maculatus (Fabr.) under a constant temperature of 29 ± 1 °C and a relative humidity of 65 ± 5% R.H. Four concentrations (0.125, 0.25, 0.5 and 1.0%) of each oil were used against the adult and tested their latent effect on the immature stages of the beetle. The results obtained show insecticidal effect of both ethyl oleate and A. calamus oil; however, A. calamus oil proved to be more toxic to the adults at all the concentrations tested in comparison to the ethyl oleate oil. As the concentration of the oils decreased, the percentage mortality decreased. The obtained results concentrated on biological efficacy of the oils tested against the adults. Both oils affected the longevities of both male and female C. maculatus adults in comparison to the control. The oviposition periods of the adult female were affected significantly in comparison to the control. A. calamus oil prohibited oviposition completely at high concentrations of 0.5 and 1.0% and caused complete sterility to the females. Lower concentrations decreased the oviposition period greatly and reduced the number of eggs laid in comparison to the control. Ethyl oleate had an inconsiderable latent effect on the incubation period as well as the percentage of hatching of the resulting eggs. A. calamus oil decreased the percentage of egg hatchability with serious increase in the percent sterility. The two tested oils had slight extension in either larval or pupal stage of the resulting progeny of C. maculatus. Both oils show oviposition deterrency towards C. maculatus adults. The oviposition deterrent index was 100% when mung bean seeds were treated with A. calamus oil. The bruchid females laid all eggs on control seeds and no eggs were laid on treated seeds.     

Characterization of resistance to three bruchid species (Callosobruchus spp., Coleoptera,Bruchidae) in cultivated rice bean (Vigna umbellata)

Resistance of wild and cultivated rice bean (Vigna umbellata [Thunberg] Ohwi and Ohashi) to three bruchid species, Callosobruchus chinensis L., Callosobruchus maculatus F., and Callosobruchus analis F., was evaluated. All but three accessions of cultivated, and all wild rice bean accessions tested, exhibited complete resistance to all three bruchid species. Rice bean seeds with seed coat removed also showed complete resistance to the three bruchid species. Results indicate that physical attributes and/or chemical(s) present in the seed coat of rice bean are not the main factors responsible for resistance. Feeding tests were performed by using artificial beans prepared with varying proportions of rice bean (resistant) and azuki bean (susceptible) flour. Number of bruchid adults that emerged decreased, and larval developmental period (days) was extended, when artificial beans with an increasing proportion of rice bean flour were used. These tests revealed that a chemical compound(s) contained in the cotyledon of rice bean has an inhibitory effect on the growth of these bruchid species. The results also indicate that the chemical(s) in rice bean cotyledon is most effective against C. maculatus.     

Use of micro-CAT scans to understand cowpea seed resistance to Callosobruchus maculatus

The cowpea bruchid, Callosobruchus maculatus (F.) (Coleoptera: Chrysomelidae: Bruchini), is a worldwide pest of stored cowpea grain [ Vigna unguiculata (L.) Walp. (Leguminosae)], causing tens of millions of dollars' worth of damage annually. One method of managing this pest involves planting cultivars whose seeds are resistant to bruchids. Despite extensive research, we still do not know the mechanism by which TVu 2027, the original resistant cowpea line, resists C. maculatus . Using micro-CAT (computerized axial tomography) scan imaging, we visualized the feeding patterns of bruchids living and growing within cowpea seeds. We present evidence that an interior zone in TVu 2027 seeds is responsible for the high mortality and developmental delays experienced by avirulent larval bruchids. We observed that both virulent as well as avirulent bruchid strains have different feeding patterns in TVu 2027 seeds compared to susceptible seeds. It appears that the resistance factor is most concentrated in a zone in each cotyledon adjacent to the air space separating the two seed halves.     

How host larval age, and nutrition and density of the parasitoid Dinarmus basalis (Hymenoptera: Pteromalidae) influence control of Acanthoscelides obtectus (Coleoptera: Bruchidae)

Choice of the targeted host developmental stage, regulation of parasitoid numbers released and introduction of food supplements are operational factors with a potential to influence the level of biological control. In a closed laboratory storage system maintained over two generations of the host, the impact of these three parameters on the control potential of the parasitoid Dinarmus basalis Rondani was investigated for high populations of larvae of Acanthoscelides obtectus (Say) feeding inside dry common bean seeds Phaseolus vulgaris. The beans were already infested with immature bruchids at the beginning of the storage period to simulate harvest conditions, characterized in a previous study. Treatments resulted in a reduction of 48-75% of the bruchid population within 16 weeks of storage. The best timing of parasitoid release was at the simulated harvest, as later releases reduced the bruchid population only by about half this percentage. Host feeding is postulated to be the key factor involved in the observed difference. The effect of increasing the number of parasitoids strongly depended on host age and food supplement. Addition of vials with honey had no direct effect on the bruchid population or on the parasitoid progeny. The ecological significance of these findings and implications for biological control are discussed.     

Talisia esculenta lectin and larval development of Callosobruchus maculatus and Zabrotes subfasciatus (Coleoptera: Bruchidae)

Bruchid larvae cause major losses in grain legume crops throughout the world. Some bruchid species, such as the cowpea weevil and the Mexican bean weevil, are pests that damage stored seeds. Plant lectins have been implicated as antibiosis factors against insects, particularly the cowpea weevil, Callosobruchus maculatus. Talisia esculenta lectin (TEL) was tested for anti-insect activity against C. maculatus and Zabrotes subfasciatus larvae. TEL produced ca. 90% mortality to these bruchids when incorporated in an artificial diet at a level of 2% (w/w). The LD(50) and ED(50) for TEL was ca. 1% (w/w) for both insects. TEL was not digested by midgut preparations of C. maculatus and Z. subfasciatus. The transformation of the genes coding for this lectin could be useful in the development of insect resistance in important agricultural crops.     

Molecular identification of four different alpha-amylase inhibitors from baru (Dipteryx alata) seeds with activity toward insect enzymes

The endophytic bruchid pest Callosobruchus maculatus causes severe damage to storage cowpea seeds, leading to economical losses. For this reason the use of alpha-amylase inhibitors to interfere with the pest digestion process has been an interesting alternative to control bruchids. With this aim, alpha-amylase inhibitors from baru seeds (Dipteryx alata) were isolated by affinity chromatographic procedures, causing enhanced inhibition of C. maculatus and Anthonomus grandis alpha-amylases. To attempt further purification, this fraction was applied onto a reversed-phase HPLC column, generating four peaks with remarkable inhibition toward C. maculatus alpha-amylases. SDS-PAGE and MALDI-ToF analysis identified major proteins of approximately 5.0, 11.0, 20.0 and 55 kDa that showed alpha-amylase inhibition. Results of in vivo bioassays using artificial seeds containing 1.0% (w/w) of baru crude extract revealed 40% cowpea weevil larvae mortality. These results provide evidence that several alpha-amylase inhibitors classes, with biotechnological potential, can be isolated from a single plant species.     

Effects of host age and host availability on developmental period,adult size,sex ratio,longevity and fecundity inLariophagus distinguendus Förster (Hymenoptera: Pteromalidae)

The effects of host age on parasitoid reproductive capacity are studied using the pteromalid parasitoid Lariophagus distinguendusFörster and its bruchid hosts, Callosobruchus chinensis (L.) and C. maculatus (F.). A series of experiments were performed to investigate relationships between age and size of host parasitized and the developmental period of pre-imaginal progeny, sex ratio, female size, longevity, fecundity and oviposition rate. There was no effect of host size on preimaginal parasitoid developmental period. Sex ratio varied from less than 5% females from young (small) hosts to 60% females from mature (large) hosts. Adult size, female longevity, fecundity, and oviposition rate were also positively related to host age. Females provided mature hosts lived longer than those provided either young hosts or no hosts, possibly because of an increased ability to host-feed from the larger hosts. The implications of these findings to parasitoid population reproductive capacity and host-parasitoid synchrony are discussed.     

Insecticidal action of Bauhinia monandra leaf lectin (BmoLL) against Anagasta kuehniella (Lepidoptera: Pyralidae), Zabrotes subfasciatus and Callosobruchus maculatus (Coleoptera: Bruchidae)

Bruchid beetle larvae cause major losses in grain legume crops throughout the world. Some bruchid species, such as the cowpea weevil (Callosobruchus maculatus) and the Mexican bean weevil (Zabrotes subfasciatus), are pests that damage stored seeds. The Mediterranean flour moth (Anagasta kuehniella) is of major economic importance as a flour and grain feeder; it is often a severe pest in flour mills. Plant lectins have been implicated as antibiosis factors against insects. Bauhinia monandra leaf lectin (BmoLL) was tested for anti-insect activity against C. maculatus, Z. subfasciatus and A. kuehniella larvae. BmoLL produced ca. 50% mortality to Z. subfaciatus and C. maculatus when incorporated into an artificial diet at a level of 0.5% and 0.3% (w/w), respectively. BmoLL up to 1% did not significantly decrease the survival of A. kuehniella larvae, but produced a decrease of 40% in weight. Affinity chromatography showed that BmoLL bound to midgut proteins of the insect C. maculatus. 33 kDa subunit BmoLL was not digested by midgut preparations of these bruchids. BmoLL-fed C. maculatus larvae increased the digestion of potato starch by 25% compared with the control. The transformation of the genes coding for this lectin could be useful in the development of insect resistance in important agricultural crops.     

The life history continuum hypothesis links traits of male ants with life outside the nest

Biological control of bruchid beetles, Callosobruchus maculatus (Fabricius) (Coleoptera: Bruchidae), infesting cowpea seeds, Vigna unguiculata (L.) Walpers (Fabaceae), can be performed via augmentative releases of Dinarmus basalis Rondani (Hymenoptera: Pteromalidae) parasitoid wasps. Females of the latter species are therefore likely to experience intense intraspecific competition: they should encounter numerous previously parasitized hosts but also conspecific competitors, with which they may fight to secure hosts on which to lay their eggs. Such contests might therefore disrupt biological control programs. Here, we studied aggressive behavior that D. basalis females show toward conspecific competitors and subsequent host exploitation strategies. We further investigated factors that classically affect contest intensity and outcomes in animals, such as the effect of ownership status, by manipulating the residency period before the intruder's arrival. In addition, we tested the effect of the size of female reproductive tissue (measured in terms of egg load) and the quality of the habitat previously experienced by females (either rich or poor in hosts). These two factors are expected to influence the value that females place on the host and therefore the costs they are willing to pay to win it. Finally, we discussed the consequences of agonistic behaviors on females' host exploitation strategies. Our results suggest that contest competition may actually enhance host control by favoring parasitoid dispersion, rather than disrupting it.     

Fumigant toxicity of plant essential oils against Camptomyia corticalis (Diptera: Cecidomyiidae)

The toxicity of 98 plant essential oils against third instars of cecidomyiid gall midge Camptomyia corticalis (Loew) (Diptera: Cecidomyiidae) was examined using a vapor-phase mortality bioassay. Results were compared with that of a conventional insecticide dichlorvos. Based on 24-h LC50 values, all essential oils were less toxic than dichlorvos (LC50, 0.027 mg/cm3). The LC50 of caraway (Carum carvi L.) seed, armoise (Artemisia vulgaris L.), clary sage (Salvia sclarea L.), oregano (Origanum vulgare L.), lemongrass [Cymbopogon citratus (DC.) Stapf], niaouli (Melaleuca viridiflora Gaertner), spearmint (Mentha spicata L.), cassia especial (Cinnamomum cassia Nees ex Blume), Dalmatian sage (Salvia offcinalis L.), red thyme (Thymus vulgaris L.), bay [Pimenta racemosa (P. Mill.) J.W. Moore], garlic (Allium sativum L.), and pennyroyal (Mentha pulegium L.) oils is between 0.55 and 0.60 mg/cm3. The LC50 of cassia (C. cassia, pure and redistilled), white thyme (T. vulgaris), star anise (Illicium verum Hook.f.), peppermint (Mentha X piperita L.), wintergreen (Gaultheria procumbens L.), cinnamon (Cinnamomum zeylanicum Blume) bark, sweet marjoram (Origanum majorana L.), Roman chamomile [Chamaemelum nobile (L.) All.], eucalyptus (Eucalyptus globulus Labill.), rosemary (Rosmarinus officinalis L.),Virginian cedarwood (Juniperus virginiana L.), pimento berry [Pimenta dioica (L.) Merr.], summer savory (Satureja hortensis L.), lavender (Lavandula angustifolia Mill.), and coriander (Coriandrum sativum L.) oils is between 0.61 and 0.99 mg/cm3. All other essential oils tested exhibited low toxicity to the cecidomyiid larvae (LC50, >0.99 mg/cm3). Global efforts to reduce the level of highly toxic synthetic insecticides in the agricultural environment justify further studies on the active essential oils as potential larvicides for the control of C. corticalis populations as fumigants with contact action.     

Fumigant toxicity of Eucalyptus intertexta, Eucalyptus sargentii and Eucalyptus camaldulensis against stored-product beetles

Abstract:  Three essential oils extracted from the species of the family Myrtaceae found in Kashan (centre regions of Iran) were shown to have potent fumigant toxicity against three major stored-product insects: Callosobruchus maculatus (Fab.), Sitophilus oryzae (L.) and Tribolium castaneum (Herbst). These were the essential oils from Eucalyptus intertexta R.T. Baker, Eucalyptus sargentii Maiden and Eucalyptus camaldulensis Dehnh. The mortality of 1- to 7-day-old adults of the insect pests increased with concentration from 37 to 926  μ l/l air and with exposure time from 3 to 24 h. The LC₅₀ values to the selected essential oils were between 2.55 and 3.97  μ l/l air for C. maculatus , 6.93 and 12.91  μ l/l for S. oryzae and 11.59 and 33.50  μ l/l air for T. castaneum . Fumigant effects of these essential oils were considered to warrant further research into their potential for commercial use.     

Toxicity of plant essential oils and their components against Lycoriella ingenua (Diptera: Sciaridae)

Plant essential oils from 20 plant species were tested for their insecticidal activity against larvae of Lycoriella ingenua (Dufour) (Diptera: Sciaridae) by using a fumigation bioassay. Good insecticidal activity (>90%) against larvae of L. ingenua was achieved with essential oils of caraway seed Carum carvi (L.)], lemongrass [Cymbopogon citratus (D.C.) Stapf.], mandarine (Citrus reticulate Blanco), nutmeg (Myristica fragrans Houtt), cade (Juniperus oxycedrus L.), spearmint (Mentha spicata L.), cumin (Cuminum cyminum L.), and thyme red [Thymus vulgaris (L.)] oils at 30 X 10-3 mg/1 air. Among them, caraway seed, spearmint, cumin, and thyme red essential oils were highly effective against L. ingenua at 20 x 10(-3) mg/ml air. Analysis by gas chromatography-mass spectrometry led to identification of 4, 9, 8, and 17 compounds from caraway seed, spearmint, cumin, and thyme red oils, respectively. These compounds were tested individually for their insecticidal activities against larvae of L. ingenua, and compared with the toxicity of dichlorvos. Carvacrol, thymol, linalool, cuminaldehyde, p-cymen, terpinen-4-ol, and carvone was effective at 10 x 10(-3) mg/l. The insecticidal activity of dichlorvos was 60% at 10 x 10(-3) mg/ml. Effects of four selected plant essential oils on growth of oyster mushroom, Pleurotus ostreatus, also were investigated.     

Presence of the storage seed protein vicilin in internal organs of larval Callosobruchus maculatus (Coleoptera: Bruchidae)

Variant vicilins (7S storage globulins) of cowpea seeds (Vigna unguiculata) are considered as the main resistance factor present in some African genotypes against the bruchid Callosobruchus maculatus. It has been suggested that the toxic properties of vicilins may be related to their recognition and interaction with glycoproteins and other membrane constituents along the digestive tract of the insect. However, the possibility of a systemic effect has not yet been investigated. The objective of this work was to study the fate of 7S storage globulins of V. unguiculata in several organs of larvae of the cowpea weevil C. maculatus. Results demonstrated binding of vicilins to brush border membrane vesicles, suggesting the existence of specific receptors. Vicilins were detected in the haemolymph, in the midgut, and in internal organs, such as fat body and malpighian tubules. There is evidence of accumulation of vicilins in the fat body of both larvae and adults. The absorption of vicilins and their presence in insect tissues parallels classical sequestration of secondary compounds.