Expression of Pinellia ternata leaf agglutinin under rolC promoter confers resistance against a phytophagous sap sucking aphid, Myzus persicae

BACKGROUND: Piercing/sucking insect pests in the order Hemiptera causes substantial crop losses by removing photoassimilates and transmitting viruses to their host plants. Cloning and heterologous expression of plantderived insect resistance genes is a promising approach to control aphids and other sap-sucking insect pests. While expression from the constitutive 35S promoter provides broad protection, the phloem-specific rolC promoter provides better defense against sap sucking insects. The selection of plant-derived insect resistance genes for expression in crop species will minimize bio-safety concerns. RESULTS: Pinellia ternata leaf agglutinin gene (pta), encodes an insecticidal lectin, was isolated and cloned under the 35S and rolC promoters in the pGA482 plant transformation vector for Agrobacterium-mediated tobacco transformation. Integration and expression of the transgene was validated by Southern blotting and qRT-PCR, respectively. Insect bioassays data of transgenic tobacco plants showed that expression of pta under rolC promoter caused 100% aphid mortality and reduced aphid fecundity up to 70% in transgenic tobacco line LRP9. These results highlight the better effectivity of pta under rolC promoter to control phloem feeders, aphids. CONCLUSIONS: These findings suggested the potential of PTA against aphids and other sap sucking insect pests. Evaluation of gene in tobacco under two different promoters; 35S constitutive promoter and rolC phloemspecific promoter could be successfully use for other crop plants particularly in cotton. Development of transgenic cotton plants using plant-derived insecticidal, PTA, would be key step towards commercialization of environmentally safe insect-resistant crops.

Pectin Methylesterases Modulate Plant Homogalacturonan Status in Defenses against the Aphid Myzus persicae.

Because they suck phloem sap and act as vectors for phytopathogenic viruses, aphids pose a threat to crop yields worldwide. Pectic homogalacturonan (HG) has been described as a defensive element for plants during infections with phytopathogens. However, its role during aphid infestation remains unexplored. Using immunofluorescence assays and biochemical approaches, the HG methylesterification status and associated modifying enzymes during the early stage of Arabidopsis (Arabidopsis thaliana) infestation with the green peach aphid (Myzus persicae) were analyzed. Additionally, the influence of pectin methylesterase (PME) activity on aphid settling and feeding behavior was evaluated by free choice assays and the Electrical Penetration Graph technique, respectively. Our results revealed that HG status and HG-modifying enzymes are significantly altered during the early stage of the plant-aphid interaction. Aphid infestation induced a significant increase in total PME activity and methanol emissions, concomitant with a decrease in the degree of HG methylesterification. Conversely, inhibition of PME activity led to a significant decrease in the settling and feeding preference of aphids. Furthermore, we demonstrate that the PME inhibitor AtPMEI13 has a defensive role during aphid infestation, since pmei13 mutants are significantly more susceptible to M. persicae in terms of settling preference, phloem access, and phloem sap drainage.

Bacterial communities of Aphis gossypii and Myzus persicae (Hemiptera: Aphididae) from pepper crops (Capsicum sp.).

Insects harbor a wide variety of microorganisms that form complex and changing communities and play an important role in the biology and evolution of their hosts. Aphids have been used as model organisms to study microorganism-insect interactions. Almost all aphids are infected with the obligate endosymbiont Buchnera aphidicola and can host different bacteria that allow them to acquire traits of agronomic importance, such as resistance to high temperatures and/or defense against natural enemies. However, the bacterial communities of most aphid species remain poorly characterized. In this study, we used high-throughput DNA sequencing to characterize the bacterial communities of Aphis gossypii and Myzus persicae from two cultivable pepper species, Capsicum frutescens (Tabasco variety) and C. annuum (Cayenne variety), in four localities of southwestern Colombia. In addition, we evaluated the dynamics of A. gossypii-associated microorganisms on a seasonal basis. Our results show that the bacterial communities of A. gossypii and M. persicae are dominated by the primary endosymbiont B. aphidicola, while the presence of the facultative symbiont Arsenophonus sp. was only detected in one A. gossypii population from cayenne pepper. In addition to these two known symbionts, eight bacterial OTUs were identified that presented a frequency of 1% or more in at least one of the analyzed populations. The results show that the bacterial communities of aphids associated with pepper crops appears to be structured according to the host aphid species and the geographical location, while no differences were observed in the diversity of bacteria between host plants. Finally, the diversity and abundance of the A. gossypii bacterial community was variable among the four sampling points evaluated over the year and showed a relation with the aphid's population dynamics. This study represents the first approach to the knowledge of the bacterial community present in chili pepper aphids from Colombia. Nevertheless, more in-depth studies, including replicates, are required to confirm the patterns observed in the microbial communities of aphids from pepper crops.

Salicylic acid-induced changes in physiological parameters and genes of the flavonoid biosynthesis pathway in Artemisia vulgaris and Dendranthema nankingense during aphid feeding.

Phloem-feeding aphids cause serious damage to plants. The mechanisms of plant-aphid interactions are only partially understood and involve multiple pathways, including phytohormones. In order to investigate whether salicylic acid (SA) is involved and how it plays a part in the defense response to the aphid Macrosiphoniella sanbourni, physiological changes and gene expression profiles in response to aphid inoculation with or without SA pretreatment were compared between the aphid-resistant Artemisia vulgaris 'Variegata' and the susceptible chrysanthemum, Dendranthema nankingense. Changes in levels of reactive oxygen species, malondialdehyde (MDA), and flavonoids, and in the expression of genes involved in flavonoid biosynthesis, including PAL (phenylalanine ammonia-lyase), CHS (chalcone synthase), CHI (chalcone isomerase), F3H (flavanone 3-hydroxylase), F3'H (flavanone 3'-hydroxylase), and DFR (dihydroflavonol reductase), were investigated. Levels of hydrogen peroxide, superoxide anions, MDA, and flavonoids, and their related gene expression, increased after aphid infestation and SA pretreatment followed by aphid infestation; the aphid-resistant A. vulgaris exhibited a more rapid response than the aphid-susceptible D. nankingense to SA treatment and aphid infestation. Taken together, our results suggest that SA could be used to increase aphid resistance in the chrysanthemum.

Ocurrencia natural de hongos entomophthorales de áfidos plaga de Medicago sativa L. en Argentina / Natural occurrence of entomophthoroid fungi of aphid pests on Medicago sativa L. in Argentina

Se encontraron cuatro especies de hongos Entomophthorales, Pandora neoaphidis, Zoophthora radicans, Entomophthora planchoniana (Entomophthorales: Entomophthoraceae) y Neozygites fresenii (Neozygitales: Neozygitaceae) infectando a Aphis craccivora, Therioaphis trifolii, Acyrthosiphon pisum y a especies no identificadas pertenecientes al género Acyrthosiphon en cultivos de alfalfa (Medicago sativa L.), en la Argentina. Los muestreos fueron realizados en cinco sitios (Ceres, Rafaela, Sarmiento, Monte Vera y Bernardo de Irigoyen) de la provincia de Santa Fe. Zoophthora radicans fue el patógeno más importante registrado principalmente en Acyrthosiphon sp. Zoophthora radicans fue exitosamente aislado y mantenido en cultivos puros. Este estudio documenta por primera vez en la Argentina la presencia de hongos Entomophthorales infectando áfidos en alfalfa

Four species of entomophthoroid fungi, Pandora neoaphidis (Entomophthorales: Entomophthoraceae), Zoophthora radicans (Entomophthorales: Entomophthoraceae), Entomophthora planchoniana (Entomophthorales: Entomophthoraceae) and Neozygites fresenii (Neozygitales: Neozygitaceae) were found to infect Aphis craccivora, Therioaphis trifolii, and Acyrthosiphon pisum and unidentified species of Acyrthosiphon on lucerne in Argentina. Samples were collected from five sites (Ceres, Rafaela, Sarmiento, Monte Vera and Bernardo de Irigoyen) in the province of Santa Fe. In this study, Zoophthora radicans was the most important pathogen and was recorded mainly on Acyrthosiphon sp. Zoophthora radicans was successfully isolated and maintained in pure cultures. This study is the first report of entomophthoroid fungi infecting lucerne (Medicago sativa L.) aphids in Argentina

Winter prevalence of obligate aphid pathogen Pandora neoaphidis mycosis in the host Myzus persicae populations in southern China: modeling description and biocontrol implication

Pandora neoaphidis overwintering had been investigated by monitoring its prevalence in Myzus persicae populations in open fields. Cabbage plants in field plots were weekly taken after mycosis initiation, to count and examine the living and dead aphids infected by P. neoaphidis. Based on the field data, infection levels (I) varied with field temperature (T), relative humidity (RH) and aphid count (numbers of living aphids per plant, N) over days (D), fitting well to the modified logistic equation I=0.91/[1+exp(8.5+(2.0H T H RH-20.2NI0)D)] (r²=0.897), where H T indicated daily hours of low temperature (<4°C), H RH daily hours of high air humidity (>90% RH) and I0 primary infection level. The model demonstrated the abiotic and biotic factors influencing P. neoaphidis mycosis development in winter, and also verifies the fungal overwintering by infecting available host aphids without a resting stage. Ultimately, P. neoaphidis mycosis reduced 81.4% of aphid populations, presenting great potential for biocontrol.

Evaluación de la virulencia de hongos entomopatógenos del pulgón del ciprés, de dos regiones ecológicas de Chile. II. / Evaluation of the virulence of entomopathogenic fungi of cypress aphid, from two ecological regions of Chile. II

El pulgón del ciprés Cinara cupressi, se considera uno de los insectos invasores más dañinos del mundo, ha provocado una gran mortalidad en especies Cupresáceas nativas y exóticas en varios países. En Chile, fue detectado el año 2003 y para el año 2008 esta plaga ya se encontraba distribuida en todo el país, afectando especies cupresáceas forestales exóticas como Cupressus macrocarpa y especies nativas, tales como, Austrocedrus chilensis (Ciprés de la Cordillera) y Fitzroya cupressoides (Alerce), que están categorizadas en el estado de conservación vulnerable y en peligro respectivamente. El área forestal de Chile ha potenciado y privilegiado el uso de controladores biológicos como parte del manejo integrado de plagas. Es por esto que se ha utilizado el parasitoide Pauesia juniperorum. Sin embargo, se han detectado bajos niveles de parasitismo. Se propone el uso de hongos entomopatógenos como alternativa y complemento al control biológico. Se prospectaron hongos entomopatógenos en la Región ecológica mediterránea per-húmeda y en la Región oceánica con influencia mediterránea. Como resultado se han identificado seis géneros de hongos descritos como patógenos de insectos. Sin embargo, bajo condiciones de laboratorio sólo cepas del género Verticillium fueron virulentas, causando sobre el 80 por ciento de mortalidad acumulada a los 7 días. No hubo diferencias significativas entre las cepas de Verticillium y un producto químico (pirimicarb) aplicado como control, aunque el hongo fue más lento. Las cepas más virulentas fueron identificadas como Verticillium lecanii, Ve 1 y Ve 2, con un TL50 de: 3.2 y 3.1 días y un DL50 : 1.24 y 1.37 conidias mL-1 respectivamente, sugiriendo el uso de estos hongos para controlar C. cupressi en Chile.

The cypress aphid, Cinara cupressi, is considered one of the most important invasive species causing high mortality in exotic and native species of Cupressaceae in several countries in the world. In Chile it was detected in 2003 and in 2008 was distributed throughout the country affecting the exotic forest species Cupressus macrocarpa and the native forest species Austrocedrus chilensis (Ciprés de la Cordillera) and Fitzroya cupressoides (Alerce), both classified as vulnerable and endangered species respectively. Efforts to their management have focused on biological control by using the parasitoid Pauesia juniperorum but until now it has not reached satisfactory control. We propose using entomopathogenic fungi, as alternative and complementary biocontrol. Entomopathogenic fungi were prospected in the ecological region Mediterranean per-humid and in the ecological region Oceanic with mediterranean influence in both colonies of C. cupressi. There were identified six genera of fungi described as insect pathogens. However, in laboratory assays only Verticillium strains were virulent, causing about 80 percent cumulative mortality at seven days. There were not significant differences among strains of Verticillium and chemical (pirimicarb) applied as control, although fungi were slower. The most virulent strains were two Verticillium lecanii, Ve 1 and Ve 2, with an LT50 of: 3.2 and 3.1 days and LD50 : 1.24 and 1.37 conidia mL-1 respectively, suggesting the use of these fungi to control C. cupressi in Chile.

Análise de sobrevivência aplicada ao desenvolvimento do pulgão Aphis gossypii Glover, 1877 (Hemiptera: Aphididae) em plantas de pepino (Cucumis sativus L. ) contaminadas com fungicidas / Survival analysis applied to the development of Aphis gossypii Glover, 1877 (Hemiptera: Aphididae) on fungicide-contaminated cucumber plants (Cucumis sativus L. )

Objetivou-se avaliar a influência de fungicidas utilizados em cultura de pepino sobre o desenvolvimento do pulgão Aphis gossypii Glover em condições de casa-de-vegetação. O delineamento experimental foi inteiramente ao acaso com quatro tratamentos e 45 repetições, sendo cada parcela composta por uma ninfa de primeiro ínstar, proveniente de uma fêmea áptera no início do período reprodutivo. Os fungicidas utilizados e suas respectivas dosagens em g i.a.L-1, foram enxofre 800 PM (1,6), mancozebe 800 PM (1,6) e oxicloreto de cobre 840 PM (1,5). A testemunha foi composta somente por água. Os produtos avaliados não influenciaram o tempo mediano de vida em cada ínstar e da fase ninfal, sendo respectivamente de 1 e 4 dias. Oxicloreto de cobre e mancozebe aumentaram o tempo mediano de vida em dias do ciclo biológico total e da fase adulta desse afídeo.

It was aimed to evaluate the influence of fungicides utilized in cucumber on the development of the aphid Aphis gossypii Glover under greenhouse conditions. The experimental design was completely randomized with four treatments and 45 replicates, being each plot consisting of a first instar nymph coming from an apterous female in early reproductive period. The fungicides utilized and their respective dosages at g i.a.L-1 were 800 PM sulphur (1.6), 800 PM mancozeb (1.6) and 840 PM copper oxichloride (1.5). The control was made up of water only. The products did not influence the median time of life in each instar and the nymphal stage, being respectively 1 and 4 days. Copper oxychloride and mancozeb increased the median time of life in days and the total life cycle of the adult stage of this species.

Prospección de hongos entomopatógenos del pulgón del ciprés en dos regiones ecológicas de Chile: I / Prospection of entomopathogenic fungi of cypress aphid in two ecological regions of Chile: I

El pulgón del ciprés (Cinara cupressi), es una plaga exótica distribuida en todo el territorio continental chileno que ha provocado daños importantes tanto enespecies forestales introducidas como nativas. Se han estudiado algunos aspectos de la biología de estepulgón, mediante control biológico y productos químicos, los cuales no presentan actualmente resultadossatisfactorios. Durante el tiempo que esta plaga se ha establecido en Chile, no se ha realizado un estudio sistemático de hongos entomopatógenos asociados aeste pulgón, aunque se cuenta con antecedentes de un control promisorio en pulgones con estos agentes enotros países. Con el objetivo inicial de aislar en una primera fase del presente estudio hongos patogénicospotenciales para el control biológico de C. cupressi, iniciamos su búsqueda en dos regiones ecológicas del sur de Chile (Región ecológica Mediterránea Per-Húmeda y Región ecológica Oceánica con influencia Mediterránea), seleccionándose en cada una de ellas 6sitios de muestreo donde en cada uno se recolectaron 10 ramas con colonias de pulgones que fueron mantenidas en una cámara bioclimática (20 +/- 2 °C, 16:8 h) por 7 días, para estimular el desarrollo fúngico. Se aislaron integrantes de lo géneros Verticillium, Paecilomyces(ambos con mayor frecuencia de presencia), Fusarium y un entomophthoral en estudio.

The cypress aphid (Cinara cupressi) is an exotic plague distributed throughout the Chilean continental territory which has caused significant damage both inintroduced forest species as well as in native ones. Some characteristics as to the biology of this aphid have beenstudied by means of biological control and chemicals yet they have not revealed any satisfactory results up tonow. During the time of occurrence of this plague in Chile systematic study on entomopathogenic fungi associated to this aphid has failed to be accomplishedalthough information about a promissory control in aphids with these agents has been reported in other countries. In order to carry out an isolation of potential pathogenic fungi for the biological control of C.cupressiin the first phase of this present research, we began to look for them in two ecological regions from southernChile (Ecological Mediterranean Per-humid Region and Ecological Oceanic Region having Mediterraneaninfluence). Six sampling sites were selected in each of them while 10 branches infected with aphid colonies were kept in a bioclimatic chamber (20+2ºC, 16:8 h) for 7 days to stimulate fungal growth. Fungi of generaVerticillium, Paecilomyces (both showing the highest occurrence frequency), Fusarium and an entomophtoral under study were isolated.

Primer Registro para el Perú de Brachycaudus schwartzi (Bórner) (Hemiptera: Aphididae) / First Report of Brachycaudus schwartzi (Bórner) from Peru (Hemiptera: Aphididae)

Se registra por primera vez para el Perú la especie Brachycaudus schwartzi (Bórner) (Hemiptera: Aphididae), "pulgón pardo del duraznero". La especie ha sido colectada en el departamento de Tacna. Material de referencia se encuentra depositado en la Colección Referencial de Insectos de la Unidad del Centro de Diagnóstico de Sanidad Vegetal del SENASA, Lima, Perú.

The species Brachycaudus schwartzi (Bórner), "brown peach aphid", is reported for the first time from Peru. This species has been collected in Tacna Department. Voucher specimens were deposited in the Colección Referencial de Insectos of the Centro de Diagnóstico de Sanidad Vegetal, SENASA, Lima, Peru.

Pre-pupation behaviour of the aphid parasitoid Aphidius ervi (Haliday) and its consequences for pre-imaginal learning.

Olfactory learning may occur at different stages of insect ontogeny. In parasitoid wasps, it has been mostly shown at adult emergence, whilst it remains controversial at pre-imaginal stages. We followed larval growth of the parasitoid wasp, Aphidius ervi Haliday, inside the host aphid, Acyrthosiphom pisum Harris, and characterised in detail the behaviour of third instar larvae. We found that just before cocoon spinning begins, the third instar larva bites a hole through the ventral side of the mummified aphid exoskeleton. We then evaluated whether this period of exposure to the external environment represented a sensitive stage for olfactory learning. In our first experiment, the third instar larvae were allowed to spin their cocoon on the host plant (Vicia faba L.) surface or on a plastic plate covering the portion of the host plant exposed to the ventral opening. Recently emerged adults of the first group showed a preference for plant volatiles in a glass Y-olfactometer, whereas no preference was found in adults of the second group. In a second experiment, during the period in which the aphid carcass remains open or is being sealed by cocoon spinning, third instar larvae were exposed for 24 h to either vanilla odours or water vapours as control. In this experiment, half of the parasitoid larvae were later excised from the mummy to avoid further exposure to vanilla. Adult parasitoids exposed to vanilla during the larval ventral opening of the mummy showed a significant preference for vanilla odours in the olfactometer, regardless of excision from the mummy. The larval behaviour described and the results of the manipulations performed are discussed as evidences for the acquisition of olfactory memory during the larval stage and its persistence through metamorphosis.

Identification of Russian wheat aphid (Homoptera: Aphididae) populations virulent to the Dn4 resistance gene.

The Russian wheat aphid, Diuraphis noxia (Mordvilko), is a serious worldwide pest of wheat and barley. Russian wheat aphid populations from Hungary, Russia, and Syria have previously been identified as virulent to D. noxia (Dn) 4, the gene in all Russian wheat aphid-resistant cultivars produced in Colorado. However, the virulence of Russian wheat aphid populations from central Europe, North Africa, and South America to existing Dn genes has not been assessed. Experiments with plants containing several different Dn genes demonstrated that populations from Chile, the Czech Republic, and Ethiopia are also virulent to Dn4. The Czech population was also virulent to plants containing the Dnx gene in wheat plant introduction PI220127. The Ethiopian population was also virulent to plants containing the Dny gene in the Russian wheat aphid-resistant 'Stanton' produced in Kansas. The Chilean and Ethiopian populations were unaffected by the antibiosis resistance in Dn4 plants. There were significantly more nymphs of the Chilean population on plants of Dn4 than on Dn6 plants at both 18 and 23 d postinfestation, and the Ethiopian population attained a significantly greater weight on Dn4 plants than on plants containing Dn5 or Dn6. These newly characterized virulent Russian wheat aphid populations pose a distinct threat to existing or proposed wheat cultivars possessing Dn4.

Feeding by the aphid Sipha flava produces a reddish spot on leaves of Sorghum halepense: an induced defense?

Feeding by the aphid Sipha flava produces a reddish spot on mature leaves of Sorghum halepense. The present work is aimed at determining whether this plant response entails induced resistance against the aphid. Old and young leaves showed the same response to aphid feeding (reddish coloration). Water-stressed plants displayed a similar reddish coloration to aphid-infested plants. This was verified by evaluation of absorbance peaks of the respective leaf extracts. Aphid fecundity was reduced on previously infested (and hence reddish colored) leaves. However, aphid fecundity was not affected on water stressed plants. Furthermore, aphid survival was not different on artificial diets containing increasing concentrations of the reddish pigment. It is concluded that the reddish spot is correlated with, but is not itself responsible for, the observed induced resistance of S. halepense against S. flava.