Controlling Myzus persicae with recombinant endophytic fungi Chaetomium globosum expressing Pinellia ternata agglutinin: using recombinant endophytic fungi to control aphids

Aims: Sap‐sucking insect pests have become the major threats to many crops in recent years; however, only a few biopesticides have been developed for controlling those pests. Here, we developed a novel pest management strategy, which uses endophytes to express anti‐pest plant lectins. Methods and Results: The fungal endophyte of Chaetomium globosum YY‐11 with anti‐fungal activities was isolated from rape seedlings. Pinellia ternata agglutinin (pta) gene was cloned into YY‐11 mediated by Agrobacterium tumefaciens. The positive transformants, as selected by antibiotic resistance, were evaluated using PCR and Western blot assay. We found that the recombinant endophytes colonized most of the crops, and the resistance of rape inoculated with recombinant endophytic fungi significantly inhibited the growth and reproduction of Myzus persicae. Conclusions: Our results showed that the recombinant endophytes expressing Pinellia ernata agglutinin (PTA) may endow hosts with resistance against sap‐sucking pests. Significance and Impact of the Study: This research may have important implications for using endophytes to deliver insecticidal plant lectin proteins to control sap‐sucking pests for crop protection.     

Genome editing for resistance against plant pests and pathogens

The conventional breeding of crops struggles to keep up with increasing food needs and ever-adapting pests and pathogens. Global climate changes have imposed another layer of complexity to biological systems, increasing the challenge to obtain improved crop cultivars. These dictate the development and application of novel technologies, like genome editing (GE), that assist targeted and fast breeding programs in crops, with enhanced resistance to pests and pathogens. GE does not require crossings, hence avoiding the introduction of undesirable traits through linkage in elite varieties, speeding up the whole breeding process. Additionally, GE technologies can improve plant protection by directly targeting plant susceptibility (S) genes or virulence factors of pests and pathogens, either through the direct edition of the pest genome or by adding the GE machinery to the plant genome or to microorganisms functioning as biocontrol agents (BCAs). Over the years, GE technology has been continuously evolving and more so with the development of CRISPR/Cas. Here we review the latest advancements of GE to improve plant protection, focusing on CRISPR/Cas-based genome edition of crops and pests and pathogens. We discuss how other technologies, such as host-induced gene silencing (HIGS) and the use of BCAs could benefit from CRISPR/Cas to accelerate the development of green strategies to promote a sustainable agriculture in the future.

     

Insecticidal activity of mixtures of Bacillus thuringiensis crystals with plant oils of Sinapis alba and Azadirachta indica

The main purpose of this study was to determine the insecticidal activity of mixtures of Bacillus thuringiensis crystalline toxins and plant substances that could contribute to create a new bioinsecticide of high efficiency. We decided to determine the toxicity of crystals of B. thuringiensis MPU B9 isolate, azadirachtin (NeemAzal), mustard oil and their mixtures against lepidopteran pests: Cydia pomonella, Spodoptera exigua and Dendrolimus pini. The interaction of the components in mixtures against insects was evaluated and their utility in plant protection was assessed. The crystals and plant substances showed synergistic effect against all insect species. The occurrence of synergism between two components depended on the ratio of their concentration in mixtures. Slight amount of one of the components added to the mixture led to enhanced toxicity of the preparation. The supplementation of mustard oil with B. thuringiensis toxins from up to 0.3% of the mixture resulted in more than twofold higher observed mortality of L1 larvae of D. pini in comparison with expected mortality. The beneficial insecticidal effect of the mustard oil and crystals mixture was also noted when crystals comprised up to 1.3% and from 7 to 20% against L1 caterpillars of C. pomonella and L3 larvae of S. exigua, respectively. The insecticidal efficient effect was achieved when mixture of B. thuringiensis crystals and azadirachtin was used against the three species of lepidopteran pests. The combinations in which components interacted synergistically against caterpillars are potentially useful for making a new bioinsecticide for controlling lepidopteran pests in plant protection.     

Spider Venom Toxin Protects Plants from Insect Attack

Many of the toxin proteins, that have been heterogeneously expressed in agricultural crops to provide resistance to insect pests, are too specific or are only mildly effective against the major insect pests. Spider venoms are a complex cocktail of toxins that have evolved specifically to kill insects. Here we show that the ω-ACTX-Hv1a toxin (Hvt), a component of the venom of the Australian funnel web spider (Hadronyche versuta) that is a calcium channel antagonist, retains its biological activity when expressed in a heterologous system. Expressed as a fusion protein in E. coli, the purified toxin fusion immobilized and killed Helicoverpa armigera and Spodoptera littoralis caterpillars when applied topically. Transgenic expression of Hvt in tobacco effectively protected the plants from H. armigera and S. littoralis larvae, with 100% mortality within 48 h. We conclude that the Hvt is an attractive and effective molecule for the transgenic protection of plants from herbivorous insects which should be evaluated further for possible application in agriculture. The authors Sher Afzal Khan and Zahid Mukhtar contributed equally to this work.     

Genetic transformation of cowpea (Vigna unguiculata L.) and stable transmission of the transgenes to progeny

Cowpeas are nutritious grains that provide the main source of protein, highly digestible energy and vitamins to some of the world's poorest people. The demand for cowpeas is high but yields remain critically low, largely because of insect pests. Cowpea germplasm contains little or no resistance to major insect pests and a gene technology approach to adding insect protection traits is now a high priority. We have adapted features of several legume and other transformation systems and reproducibly obtained transgenic cowpeas that obey Mendelian rules in transmitting the transgene to their progeny. Critical parameters in this transformation system include the choice of cotyledonary nodes from developing or mature seeds as explants and a tissue culture medium devoid of auxins in the early stages, but including the cytokinin BAP at low levels during shoot initiation and elongation. Addition of thiol-compounds during infection and co-culture with Agrobacterium and the choice of the bar gene for selection with phosphinothricin were also important. Transgenic cowpeas that transmit the transgenes to their progeny can be recovered at a rate of one fertile plant per thousand explants. These results pave the way for the introduction of new traits into cowpea and the first genes to be trialled will include those with potential to protect against insect pests.     

Linear transgene constructs lacking vector backbone sequences generate transgenic rice plants which accumulate higher levels of proteins conferring insect resistance

Biolistic transformation was used to introduce genes encoding the insecticidal proteins snowdrop lectin (Galanthus nivalis agglutinin; GNA) and cry1Ac Bt toxin (-endotoxin from Bacillus thuringiensis) into elite rice (Oryza sativa) cultivars. Plant transformation was carried out in parallel experiments simultaneously by using either whole plasmids containing suitable gene constructs, or the corresponding minimal gene cassettes, which were linear DNA fragments lacking vector sequences excised from the plasmids. Both transformation methods generated similar numbers of independent transformation events. Selected R₀ clonal plant lines were further characterised for presence and expression of transgenes. Co-transformation of the unselected genes (cry1Ac and gna) with the selectable marker (hpt) was at least as efficient for transformation with minimal gene cassettes as with whole plasmid DNA, and higher levels of accumulation of the insecticidal gene products GNA and cry1Ac were observed in plants resulting from minimal gene cassette transformation. Insect bioassays with major pests of rice showed that transgenic plants expressing gna showed enhanced resistance to brown planthopper (Nilaparvata lugens), and plants expressing cry1Ac were protected against attack by striped stem borer (Chilo suppressalis). Expression of both transgenes gave protection against both pests, but did not increase protection against either pest significantly over the levels observed in plants containing a single insecticidal transgene.     

A selective insecticidal protein from Pseudomonas mosselii for corn rootworm control

The coleopteran insect western corn rootworm (WCR, Diabrotica virgifera virgifera) is an economically important pest in North America and Europe. Transgenic corn plants producing Bacillus thuringiensis (Bt) insecticidal proteins have been useful against this devastating pest, but evolution of resistance has reduced their efficacy. Here, we report the discovery of a novel insecticidal protein, PIP‐47Aa, from an isolate of Pseudomonas mosselii. PIP‐47Aa sequence shows no shared motifs, domains or signatures with other known proteins. Recombinant PIP‐47Aa kills WCR, two other corn rootworm pests (Diabrotica barberi and Diabrotica undecimpunctata howardi) and two other beetle species (Diabrotica speciosa and Phyllotreta cruciferae), but it was not toxic to the spotted lady beetle (Coleomegilla maculata) or seven species of Lepidoptera and Hemiptera. Transgenic corn plants expressing PIP‐47Aa show significant protection from root damage by WCR. PIP‐47Aa kills a WCR strain resistant to mCry3A and does not share rootworm midgut binding sites with mCry3A or AfIP‐1A/1B from Alcaligenes that acts like Cry34Ab1/Cry35Ab1. Our results indicate that PIP‐47Aa is a novel insecticidal protein for controlling the corn rootworm pests.     

Incidence and Geographical Distribution of Honeybee (Apis mellifera L.) Pests in Jordan

The occurrence and distribution of pests of honeybee (Apis mellifera L.) in Jordan were surveyed during the agricultural seasons of 1999 and 2000. A total of 800 colonies in 112 apiaries were chosen from different geographical regions that support beekeeping in Jordan. Honeybee colonies are attacked by 21 pest species in different areas of Jordan. The most distributed pests which cause considerable damage are Varroa destructor, Vespa orientalis, Galleria melonella, Dorylus fulvus, Braula orientalis, Merops apiaster, Hirundo rustica, Hirundo daurica and Merops orientalis. Other pests of minor importance to beekeeping were also recorded. Most of the recorded pests were appeared in both the Jordan Valley and Irbid regions.     

A nematode,fungus, and aphid interact via a shared host plant: implications for soybean management

Soybean, Glycine max (L.) Merrill (Fabaceae), is an introduced crop to America and initially benefited from a small number of pests threatening its production. Since its rapid expansion in production beginning in the 1930s, several pests have been introduced from the native range of soybean. Our knowledge of how these pests interact and the implications for management is limited. We examined how three common economic soybean pests, the nematode Heterodera glycines Ichinohe (Nematoda: Heteroderidae), the fungus Cadophora gregata Harrington & McNew (Incertae sedis), and the aphid Aphis glycines Matsumura (Hemiptera: Aphididae), interact on soybean cyst nematode‐susceptible (SCN‐S) and soybean cyst nematode‐resistant cultivars carrying the PI 88788 resistance source (SCN‐R). From 2008 to 2010, six soybean cultivars were infested with either a single pest or all three pests in combination in a micro‐plot field experiment. Pest performance was measured in a ‘single pest’ treatment and compared with pest performance in the ‘multiple pest’ treatment, allowing us to measure the impact of SCN resistance and the presence of other soybean pests on each pest’s performance. Performance of H. glycines (80% reduction in reproduction) and A. glycines (19.8% reduction in plant exposure) was reduced on SCN‐R cultivars. Regardless of cultivar, the presence of multiple pests significantly decreased the performance of A. glycines, but significantly increased H. glycines performance. The presence of multiple pests decreased the performance of C. gregata on SCN‐S soybean cultivars (20.6% reduction in disease rating).     

Behavioural responses of Oryzaephilus surinamensis, Tribolium castaneum and Tribolium confusum to naturally and artificially damaged durum wheat kernels

Observations on behavioural activity involved in the host selection by secondary pests of stored grains, Oryzaephilus surinamensis (Linnaeus), Tribolium castaneum (Herbst) and Tribolium confusum J. du Val, with respect to intact and mechanically or naturally damaged kernels are reported. Our results indicate that the attraction of secondary pests is facilitated by broken grain kernels, which resulted from either mechanical damage during harvesting and/or binning procedures, or the feeding activity of primary insect pests. Insect damaged kernels were more attractive to O. surinamensis, T. castaneum and T. confusum than whole kernels; in addition insect damaged kernels elicit more attractiveness than mechanically split kernels. The damage caused by primary pests, such as Rhyzopertha dominica (Fabricius) and Sitophilus oryzae (Linnaeus), on whole kernels may facilitate colonization by secondary pests, which continue damaging the cereals. O. surinamensis, T. castaneum and T. confusum utilize the grain volatile odours to distinguish whether the grain kernels of the stored cereals are damaged mechanically or by insects.     

An appetite for pests: Synanthropic insectivorous bats exploit cotton pest irruptions and consume various deleterious arthropods

Conservation biological control (CBC) seeks to minimize the deleterious effects of agricultural pests by enhancing the efficiency of natural enemies. Despite the documented potential of insectivorous bats to consume pests, many synanthropic bat species are still underappreciated as beneficial species. We investigated the diet of Kuhl's pipistrelle (Pipistrellus kuhlii), a common synanthropic insectivorous bat that forages in urban and agricultural areas, to determine whether it may function as a natural enemy in CBC. Faecal samples of P. kuhlii were collected throughout the cotton‐growing season from five roost sites near cotton fields located in a Mediterranean agroecosystem, Israel, and analyzed using DNA metabarcoding. Additionally, data on estimated abundance of major cotton pests were collected. We found that the diet of P. kuhlii significantly varied according to sites and dates and comprised 27 species of agricultural pests that were found in 77.2% of the samples, including pests of key economic concern. The dominant prey was the widespread cotton pest, the pink bollworm, Pectinophora gossypiella, found in 31% of the samples and in all the roosts. Pink bollworm abundance was positively correlated with its occurrence in the bat diet. Furthermore, the bats’ dietary breadth narrowed, while temporal dietary overlap increased, in relation to increasing frequencies of pink bollworms in the diet. This suggests that P. kuhlii exploits pink bollworm irruptions by opportunistic feeding. We suggest that synanthropic bats provide important pest suppression services, may function as CBC agents of cotton pests and potentially contribute to suppress additional deleterious arthropods found in their diet in high frequencies.     

Yield loss at the different growth stages in soybean due to insect pests in Ghana

Insecticide protection at the vegetative, reproductive or both vegetative and reproductive (complete) crop growth stages and untreated control was used to assess yield loss due to insect pests at the different growth stages of soybean in Ghana from 2007–2009. The objectives were to determine the economic importance of the two major insect pest guilds in soybean, viz. defoliators and pod feeders, and when to apply control measures for maximum benefit. The defoliators recorded were Podagrica spp., Ootheca mutabilis (Shalberg), Zonocerus variegatus L., Sylepta derogata F., Spodoptera littoralis Boisduval, Amsacta spp. and Helicoverpa armigera Hübner. The pod feeders recorded were the pod-sucking bugs (PSBs) Riptortus dentipes F., Thyanta sp. Aspavia armigera F., Nezara viridula L. and Dysdercus völkeri Schmidt. Generally, insect densities, pod and seed damage were lower while seed yields were significantly greater and similar in plots that were protected at the reproductive stage against PSBs and those protected at both vegetative and reproductive stages. Yield loss ranged between 25.8 and 42.8% in untreated plots, 11.1 and 34.3% in plots that were protected at the vegetative stage, and 5.2 and 11.3% in plots that were protected at the reproductive stage. There was a consistent negative correlation between yield and numbers of PSBs as well as pod and seed damage. These results showed that PSBs that attack soybean at the reproductive stage were the most important insect pests limiting soybean yield in Ghana.     

The interaction of soybean aphids and soybean cyst nematodes on selected resistant and susceptible soybean lines

Soybean aphids, Aphis glycines Matsumura, and soybean cyst nematodes, Heterodera glycines Ichinohe, are economic pests of soybean, Glycine max (L.) Merr., in the north‐central United States. Combined, these pests may account for 20–50% of yield reductions in a soybean crop. Only limited information is available concerning the interaction of these two pests on soybean production. During the summers of 2006 and 2007, we conducted a field‐experiment near Urbana, IL, to evaluate the effect of resistant and susceptible soybean lines on the development and reproduction of both pests in combination with each other. We also examined how each pest, as well as their interaction, affected the yield of susceptible and resistant soybean lines. Soybean plants grown within caged plots were infested with soybean aphids and soybean cyst nematodes; cumulative aphid days and soybean cyst nematode egg densities were determined at the end of each growing season. Soybean aphids were able to survive on all four soybean lines in both years of this study; however, aphid‐resistant lines generally had fewer cumulative aphid days than aphid‐susceptible lines. Likewise, nematode‐resistant lines typically had fewer eggs than nematode‐susceptible lines. During both years, we failed to observe a significant interaction between these two pests on the reproduction of one another. Yield data from 2006 was inconclusive; however, results from 2007 suggest that yield‐loss when soybean aphids and soybean cyst nematodes occur jointly is not significantly greater than when these two pests occur independently. The relationship between these two pests, and our inability to observe an interaction, are discussed.     

入境台湾果蔬3种重要有害生物的危害与防控

围绕入境台湾果蔬涉及的重要有害生物,在收集文献资料基础上,从传入和扩散、传入后的危害以及在中国大陆的适生区预测等3个方面阐述了橘小实蝇、三叶草斑潜蝇和螺旋粉虱在中国大陆的灾变过程。橘小实蝇、三叶草斑潜蝇和螺旋粉虱等3种有害生物相继传入台湾之后,分别经历了22年、17年和8年时间又相继传入中国大陆,且两者相距时间有缩短的趋势。橘小实蝇和三叶草斑潜蝇传入大陆后,得到迅速扩散蔓延,给大陆果蔬生产和贸易造成重大影响;而螺旋粉虱目前分布局限在海南,并在局部地区造成较严重的危害。台湾发生的有害生物在中国大陆均有较大范围的适生区,即便是已传入数十年的橘小实蝇,因气候等条件的变化,其在中国适生区也在扩大之中。根据分析结果,从有害生物风险分析、口岸查验和检疫、入侵害虫的监测与防控等方面提出有效管控入境台湾果蔬有害生物的对策措施。     

Managing Insect Resistance to Plants Producing Bacillus thuringiensis Toxins

ABSTRACT:?

Insect-resistant transgenic plants have become an important tool for the protection of crops against insect pests. The acreage of insecticidal transgenic plants is expected to increase significantly in the near future. The bacterium Bacillus thuringiensis is currently the source of insecticidal proteins in commercial insect-resistant transgenic plants and will remain the most important source during the next decade. Insect resistance to B. thuringiensis Cry toxins is the main problem. Only one species, the diamondback moth, has evolved a resistance to B. thuringiensis-based formulations under field conditions. However, many other insect species were selected for resistance under laboratory conditions, indicating that there is a potential for evolution of resistance in most major pests. Many studies were conducted to elucidate the mode of action of the Cry toxins, the mechanisms and genetics of resistance, and the various factors influencing its development. This article reviews insect resistance to B. thuringiensis insecticidal proteins and related aspects, including the development of insect-resistant transgenic plants, B. thuringiensis toxins, their mode of action, mechanisms, stability, and genetics of resistance and management strategies for delaying resistance.     

Carabid beetle condition,reproduction and density in winter oilseed rape affected by field and landscape parameters

The carabid beetles Amara similata and Poecilus cupreus are abundant in Central European winter oilseed rape (OSR) fields and potential antagonists of OSR pests. Therefore, they were investigated in 29 OSR fields relative to the influence of field and landscape parameters on their nutritional condition, reproductive potential and activity density. Nutritional condition was measured by a condition factor (CF). Fecundity of female beetles was expressed by the number of ripe oocytes in the ovaries. Activity density comprised the number of individuals caught with pitfall traps during their peak of reproductive period. Soil productivity and pest abundance (as a proxy of local prey availability) were considered as field parameters. Landscape parameters comprised the distance to the next fallow and the percentage of crop area around each study field. In eight of ten cases, pest abundance proved to be the most important factor explaining carabid characteristics, indicating that P. cupreus and A. similata consume insect pests in OSR fields. Pest abundance influenced the CF of both species and sexes positively. Oocyte numbers of A. similata were negatively related to the distance to the next fallow. Oocyte numbers of P. cupreus tended to increase with increasing pest abundance. Activity density of female A. similata was negatively influenced by soil productivity. Activity density of male A. similata and of female and male P. cupreus was negatively influenced by pest abundance. When analysing exclusively the influence of severe OSR pests (Meligethes aeneus, Ceutorhynchus napi and Dasineura brassicae), abundance of larval M. aeneus and C. napi was most important in explaining the CF and activity density of male A. similata, and male and female P. cupreus. M. aeneus and C. napi could be an essential prey for the carabids studied which may reduce the pests, thereby contributing to the protection of OSR from pest herbivory.     

Expression and purification of a novel mannose-binding lectin from Pinellia ternata

Pinellia ternata agglutinin (PTA) from the tubers of P. ternata is a monocot mannose-binding lectin that catalytically agglutinated rabbit erythrocytes. The potential effect of PTA has gained considerable interest in recent years owing to clinical use of native PTA as the preparation against cancer and for plant protection against insect pests. Here we report a successful strategy to allow high-level expression of PTA as inclusion bodies in Escherichia coli M15. Purification of refolded recombinant protein from solubilized inclusion bodies by Ni-NTA agarose affinity chromatography yielded biological activity recombinant PTA (final yield of about 10 mg/L). The recombinant PTA agglutinated rabbit erythrocytes to a dilution similar to that determined for “native” lectin purified from P. ternata. The expression and purification system makes it possible to obtain sufficient quantities of biologically active and homogenous recombinant PTA sufficient to carry out advanced clinical trials. This is the first report on the large-scale expression and purification of biologically active recombinant PTA from E. coli.     

An investigation on Lecanicillium muscarium as a biocontrol agent of stem borer pests of Rosa damascena in Kerman Province of Iran

The Rosa damascena has organic production and this plant is the most important economic crop in Kerman province. Roses have been used since the earliest times in rituals, cosmetics, perfumes, medicines and aromatherapy. The rose stem sawfly (Hartigia trimaculata) and rose stem girdler (Agrilus aurichalceus) are new and major pests of R. damascena in the Lalehzar region of Kerman province. These pests cause severe damage to plants by feeding stems and new management strategies for their control are continually being investigated. To investigate appropriate biological control agent in the region during 2005–2007, 184 isolates of fungi were collected from these pests. Isolation of fungi was achieved using standard methods. In this study, H. trimaculata and A. aurichalceus from R. damascena for the first time were recorded in Iran. Lecnicillium muscarium from H. trimaculata, L. muscarium from A. aurichalceus, Acremonium kiliense from H. trimaculata, and A. egyptiacum from A. aurichalceus have for the first time been recorded, and L. muscarium has been introduced as a suitable biological agent for control of these pests.     

<Emphasis Type="Italic">In vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> inhibition of α-amylases of stored-product mite <Emphasis Type="Italic">Acarus siro</Emphasis>

The stored-product mites are the most abundant and frequent group of pests living on the stored food products in Europe. They endanger public health since they produce allergens and transmit mycotoxin-producing fungi. Novel acaricidal compounds with inhibitory effects on the digestive enzymes of arthropods are a safe alternative to the traditional neurotoxic pesticides used for control of the stored-product pests. In this work, we explored the properties of acarbose, the low molecular weight inhibitor of -amylases (AI), as a novel acaricide candidate for protection of the stored products from infestation by Acarus siro (Acari: Acaridae). In vitro analysis revealed that AI blocked efficiently the enzymatic activity of digestive amylases of A. siro, and decreased the physiological capacity of mites gut in utilizing a starch component of grain flour. In vivo experiments showed that AI suppressed the population growth of A. siro. The mites were kept for three weeks on experimental diet enriched by AI in concentration range of 0.005 to 0.25%. Population growth of A. siro was negatively correlated with the content of AI in the treated diet with a half-population dose of 0.125%. The suppressive effect of AIs on stored-product mites is discussed in the context of their potential application in GMO crops     

The omnivorous predator Macrolophus pygmaeus,a good candidate for the control of both greenhouse whitefly and poinsettia thrips on gerbera plants

The poinsettia thrips Echinothrips americanus Morgan is a relatively new pest that has spread rapidly worldwide and causes serious damage in both vegetable and ornamental plants. In this study, we investigated if and how effective this pest can be controlled in gerbera by the omnivorous predator Macrolophus pygmaeus (Rambur). Because herbivores on plants can interact through a shared predator, we also investigated how poinsettia thrips control is affected by the presence of the greenhouse whitefly Trialeurodes vaporariorum (Westwood), a pest that commonly coexists with E. americanus in gerbera. In laboratory studies, we found that the predator M. pygmaeus fed on both pests when offered together. Olfactometer tests showed a clear preference of the predators for plants infested by whiteflies but not by thrips. In a greenhouse experiment, densities of both pests on single gerbera plants were reduced to very low levels by the predator, either with both pests present together or alone. Hence, predator‐mediated effects between whiteflies and thrips played only a minor role. The plant feeding of the shared predator probably reduced the dependence of predator survival and reproduction on the densities of the two pests, thereby weakening potential predator‐mediated effects. Thus, M. pygmaeus is a good candidate for biological control of both pests in gerbera. However, further research is needed to investigate pest control at larger scales, when the pests can occur on different plants.