Development of a model system to assess the impact of genetically modified corn and aubergine plants on arbuscular mycorrhizal fungi

We developed an experimental model system to monitor the impact of generically modified (GM) plants on arbuscular mycorrhizal (AM) fungi, a group of non-target soil microorganisms, fundamental for soil fertility and plant nutrition. The system allowed us to study the effects of root exudates of both commercial Bt corn and aubergine plants expressing Dm-AMP1 defensin on different stages of the life cycle of the AM fungal species G. mosseae. Root exudates of Bt 176 corn significantly reduced pre-symbiotic hyphal growth, compared to Bt 11 and non-transgenic plants. No differences were found in mycelial growth in the presence of Dm-AMP1 and control plant root exudates. Differential hyphal morphogenesis occurred irrespective of the plant line, suggesting that both exuded Bt toxin and defensin do not interfere with fungal host recognition mechanisms. Bt 176 affected the regular development of appressoria, 36% of which failed to produce viable infection pegs. Our experimental model system represents an easy assay for testing the impact of GM plants on non-target soil-borne AM fungi.     

Anatomical Injury Induced by the Eriophyid Mite Aceria anthocoptes on the Leaves of Cirsium arvense

Anatomical injury of the leaves of the invasive species, Cirsium arvense (L.) Scop., caused by the eriophyid mite Aceria anthocoptes (Nal.), which is the only eriophyid mite that has been recorded on C. arvense worldwide, is described. The injury induced by the mite feeding on the leaves of C. arvense results in visible russeting and bronzing of the leaves. Other conspicuous deformations are folding and distortion of the leaf blade and curling of leaf edge, as well as gradual drying of leaves. The anatomical injury of the mature leaves of field-collected plants was limited to the epidermis of the lower leaf surface. However, on young leaves of experimentally infested plants, rust mite injuries extend to epidermal cells on both leaf surfaces and to those of deeper mesophyll layers. On these leaves, lesions on the lower leaf surface even affected the phloem of the vascular bundles. Leaf damage induced by A. anthocoptes is discussed with regard to the mite’s potential as a biological control agent of C. arvense.     

Culturable endophytic filamentous fungi from leaves of transgenic imidazolinone-tolerant sugarcane and its non-transgenic isolines

The diversity of endophytic filamentous fungi from leaves of transgenic imidazolinone-tolerant sugarcane plants and its isoline was evaluated by cultivation followed by amplified rDNA restriction analysis (ARDRA) of randomly selected strains. Transgenic and non-transgenic cultivars and their crop management (herbicide application or manual weed control) were used to assess the possible non-target effects of genetically modified sugarcane on the fungal endophytic community. A total of 14 ARDRA haplotypes were identified in the endophytic community of sugarcane. Internal transcribed spacer (ITS) sequencing revealed a rich community represented by 12 different families from the Ascomycota phylum. Some isolates had a high sequence similarity with genera that are common endophytes in tropical climates, such as Cladosporium, Epicoccum, Fusarium, Guignardia, Pestalotiopsis and Xylaria. Analysis of molecular variance indicated that fluctuations in fungal population were related to both transgenic plants and herbicide application. While herbicide applications quickly induced transient changes in the fungal community, transgenic plants induced slower changes that were maintained over time. These results represent the first draft on composition of endophytic filamentous fungi associated with sugarcane plants. They are an important step in understanding the possible effects of transgenic plants and their crop management on the fungal endophytic community.     

Effects of soybean proteinase inhibitor on development, survival and reproductive potential of the sugarcane borer, Diatraea saccharalis

One approach that can be employed in integrated pest management is the use of proteins with antinutritional effects on insect metabolism and development. The antimetabolic properties of soybean proteinase inhibitor (SPI) on growth of neonate larvae of the sugarcane borer, Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae) have been evaluated. When incorporated into an artificial diet at 0.5% (w/w), SPI retarded growth rate and development of larvae when compared with larvae fed on artificial diet alone. However, larval survival was not significantly affected. The purpose of our research was to calculate demographic statistics for the sugarcane borer reared on diet either with or without semi-purified extract of SPI. Net reproductive rate (R ₀), instantaneous rate of increase (r _m), combined age-specific survivorship (l _x) and age specific fecundity (m _x) provide information about population growth potential. These parameters were measured in order to determine the effects of the proteinase inhibitor on the insect's population dynamics. The observed differences would potentially translate into large reductions in population growth, indicating a potential value of using SPI for protecting sugarcane plants against damage by the sugarcane borer.     

Induced Response of Tomato Plants to Injury by Green and Red Strains of Tetranychus Urticae

We studied the induced response of tomato plants to the green strain and the red strain of the spider mite Tetranychus urticae. We focused on the olfactory response of the predatory mite Phytoseiulus persimilis to volatiles from T. urticae-infested tomato leaves in a Y-tube olfactometer. Tomato leaves attracted the predatory mites when slightly infested with the red strain, or moderately or heavily infested with the green strain. In contrast, neither leaves that were slightly infested with green-strain mites, nor leaves that were moderately or heavily infested with the red strain attracted the predators. We discuss the specific defensive responses of tomato plants to each of the two strains.     

Field assessment of host plant specificity and potential effectiveness of a prospective biological control agent, Aceria salsolae, of Russian thistle, Salsola tragus

The eriophyid mite, Aceria salsolae de Lillo and Sobhian, is being evaluated as a prospective classical biological control agent of invasive alien tumbleweeds, including Salsola tragus, S. collina, S. paulsenii and S. australis, in North America. Previous laboratory experiments to determine the host specificity of the mite indicated that it could sometimes persist and multiply on some nontarget plants, including Bassia hyssopifolia and B. scoparia. These are both European plants whose geographic range overlaps that of the mite, but the mite has never been observed on them in the field. A field experiment was conducted in Italy to determine if the mite would infest and damage these plants under natural outdoor conditions. The results indicate that this mite does not attain significant populations on these nontarget plants nor does it significantly damage them. Salsola tragus was heavily infested by A. salsolae, and plant size was negatively correlated to the level of infestation. Although S. kali plants were also infested, their size did not appear to be affected by the mites. The other nontarget plants were not as suitable for the mite in the field as in previous laboratory experiments. We conclude that there would be no significant risk to nontarget plants as a result of using A. salsolae as a biological agent to control Salsola species in North America.     

Effects of Chilling Temperature on the Activity of Enzymes of Sucrose Synthesis and the Accumulation of Saccharides in Leaves of Three Sugarcane Cultivars Differing in Cold Sensitivity

The effects of short-term exposure to chilling temperature (10 °C) on sucrose synthesis in leaves of the cold-tolerant sugarcane cultivars Saccharum sinense R. cv. Yomitanzan and Saccharum sp. cv. NiF4, and the cold-sensitive cultivar S. officinarum L. cv. Badila were studied. Plants were grown at day/night temperatures of 30/25 °C, and then shifted to a constant day/night temperature of 10 °C. After 52-h exposure to the chilling temperature, sucrose content in the leaves of NiF4 and Yomitanzan showed a 2.5- to 3.5-fold increase relative to that of the control plants that had been left on day/night temperatures of 30/25 °C. No such increase was observed in Badila leaves. Similarly, starch content in the leaves of NiF4 and Yomitanzan was maintained high, but starch was depleted in Badila leaves after the 52-h exposure. During the chilling temperature, sucrose phosphate synthase (SPS; E.C.2.4.1.14) activity was relatively stable in the leaves of NiF4 and Yomitanzan, whereas in Badila leaves SPS activity significantly decreased. There was no significant change in cytosolic fructose-1,6-bisphosphatase activity for the three cultivars at the chilling temperature. This supports the hypothesis that: (1) on exposure to chilling temperature, sucrose content in sugarcane leaves is determined by the photosynthetic rate in the leaves, and is not related to SPS activity; (2) SPS activity in sugarcane leaves at chilling temperature is to be determined by sugar concentration in the leaves.     

A new mite-plant association: mites living amidst the adhesive traps of a carnivorous plant

Non-antagonistic interactions between arthropods and leaves of insectivorous plants with adhesive traps so far have never been reported. The mites are common prey of such plants, but we have found a new subspecies of the mite Oribatula tibialis living on the leaves of Pinguicula longifolia. Because of its small size and the low glandular density of the host, the mite moves without being trapped by the mucilaginous droplets of the leaf surface. P. longifolia provides shelter and food for the mite, while the plant may also benefit because of its fungivorous and scavenging activities. This new interaction is another dramatic example of widespread miteplant associations.     

The role of sinigrin in host plant recognition by aphids during initial plant penetration

Plant penetration behaviour (probing) of the cabbage aphid, Brevicoryne brassicae, and the pea aphid, Acyrthosiphon pisum, was studied on excised leaves of broad beans, Vicia faba, kept in water or in a 1% aqueous solution of sinigrin. Using the DC EPG (Electrical Penetration Graph) technique it was shown that the cabbage aphid on sinigrin-untreated bean leaves showed numerous short probes into epidermis and mesophyll. None of these aphids showed either phloem salivation or ingestion waveforms on untreated leaves. In contrast, on sinigrin-treated bean leaves, 35% of the probing time was spent on phloem sap ingestion (E2) and almost all aphids reached phloem vessels and started feeding. The duration of phloem salivation before phloem ingestion and the mean duration of phloem ingestion periods were similar on a host and a sinigrin-treated non-host plant. However, the total probing time by B. brassicae was 10% longer, the total phloem sap ingestion time was twice as long, and the time to the first phloem phase within a probe was three times shorter on the host plant compared to sinigrin-treated broad beans. Acyrthosiphon pisum also responded to the addition of sinigrin to broad beans, but in this case sinigrin acted as a deterrent. On sinigrin-treated leaves, A. pisum terminated probes before ingestion from phloem vessels, and none of these aphids showed phloem salivation and ingestion on treated leaves. Glucosinolates were detected in the mesophyll cells of the brassicaceous plant, Sinapis alba. Based on this finding and in addition to the foregoing EPG analysis of aphid probing on these plants and broad beans, our hypothesis is that aphids may recognise their host plants as soon as they probe the mesophyll tissue and before they start ingestion from phloem vessels.     

Comparative toxicity of some acaricides to the predatory mite, Phytoseiulus persimilis and the twospotted spider mite, Tetranychus urticae

The relative toxicity of someacaricides to the predatory mite, Phytoseiulus persimilis and the twospottedspider mite, Tetranychus urticae (Acari: Phytoseiidae, Tetranychidae) wasevaluated in laboratory. Five of theacaricides tested, including bifenazate,acequinocyl, chlorfenapyr, flufenoxuron andfenbutatin oxide, were much less toxic to adultfemales and immatures of P. persimilisthan to those of T. urticae, and adultfemale predators treated with these fiveacaricides produced 84±96% as many eggs as didcontrol females. Etoxazole did not seriouslyaffect the survival and reproduction of adultfemale predators but caused high mortalityrates in eggs and larvae of P.persimilis. Milbemectin and fenazaquin werevery toxic to adult females and immatures ofP. persimilis. Adult female predatorssurvived on a diet of spider mites treated withbifenazate, acequinocyl, chlorfenapyr,flufenoxuron and fenbutatin oxide, and theirfecundity, prey consumption and the sex ratioof the progeny were not substantially affected. Based on the results, bifenazate, acequinocyl,chlorfenapyr, flufenoxuron and fenbutatin oxideappeared to be the promising candidates for usein integrated mite management programs whereP. persimilis is the major naturalenemy.     

Long-term effects of soybean protease inhibitors on digestive enzymes, survival and learning abilities of honeybees

We have investigated the effects of long-term ingestion of two serine proteinase inhibitors (PIs), the Kunitz Soybean trypsin inhibitor (SBTI) and the Bowman-Birk inhibitor (BBI) on survival, learning abilities involved in the foraging behaviour, and digestive physiology of the honeybee (Apis mellifera L., Hymenoptera). A threshold-dose was established, above which adverse effects of long-term ingestion of the PIs tested are to be expected. The experiments reported herein could be extended to other PIs or gene products used to confer insect resistance, and be part of a general procedure used to assess the innocuousness of transgenic melliferous plants to honeybees.     

Response of predatory mites with different rearing histories to volatiles of uninfested plants

The behavioural response of the predatory mite Phytoseiulus persimilis to volatiles from several host plants of its prey, spider mites in the genus Tetranychus, was investigated in a Y-tube olfactometer. A positive response to volatiles from tomato leaves and Lima bean leaves was recorded, whereas no response was observed to volatiles from cucumber leaves, or leaves of Solanum luteum and Solanum dulcamara.Different results were obtained for predators that differed in rearing history. Predators that were reared on spider mites (Tetranychus urticae) on Lima bean leaves did respond to volatiles from Lima bean leaves, while predators that had been reared on the same spider mite species but with cucumber as host plant did not respond to Lima bean leaf volatiles. This effect is compared with the effect of rearing history on the response of P. persimilis to volatile allelochemicals of prey-infested plant leaves.     

The spatial and temporal distribution of predatory and phytophagous mites in field-grown strawberry in the UK

Extensive sampling of strawberry plants in everbearing and June-bearing strawberry plantations and on potted plants showed that different species of mites were spatially separated. Of the two phytophagous species recorded, Tetranychus urticae was most abundant on old leaves and Phytonemus pallidus on folded leaves and flower/fruit clusters. Predatory phytoseiid mites were found on all plant parts but different species were spatially separated; Neoseiulus cucumeris and N. aurescens were found mostly on folded leaves and clusters, and N. californicus and Phytoseiulus persimilis on old and medium aged leaves. No Typhlodromus pyri were found in the field plantations. These patterns of distribution did not change over sampling dates in summer and early autumn. An understanding of this within-plant zonation of mite species is important when studying predator–prey interactions and when designing sampling strategies for strawberry. A programme to sample the entire mite system on strawberry should be stratified to include all the above mentioned parts of the plant. Different sampling protocols, as appropriate, are required for sampling different pest species and their associated predators.     

Toxicological evaluation of genetically modified cotton (Bollgard<Superscript>®</Superscript>) and Dipel<Superscript>®</Superscript> WP on the non-target soil mite <Emphasis Type="Italic">Scheloribates praeincisus</Emphasis> (Acari: Oribatida)

Insecticides derived from the bacterium Bacillus thuringiensis (Bt) and plants genetically modified (GM) to express B. thuringiensis toxins are important alternatives for insect pest control worldwide. Risk assessment of B. thuringiensis toxins to non-target organisms has been extensively studied but few toxicological tests have considered soil invertebrates. Oribatid mites are one of the most diverse and abundant arthropod groups in the upper layers of soil and litter in natural and agricultural systems. These mites are exposed to the toxic compounds of GM crops or pesticides mainly when they feed on vegetal products incorporated in the soil. Although some effects of B. thuringiensis products on Acari have been reported, effects on oribatid mites are still unknown. This study investigated the effects of the ingestion of Bt cotton Bollgard and of the B. thuringiensis commercial product Dipel WP on the pantropical species Scheloribates praeincisus (Scheloribatidae). Ingestion of Bollgard and Dipel did not affect adult and immature survivorship and food consumption (estimated by number of fecal pellets produced daily) or developmental time of immature stages of S. praeincisus. These results indicate the safety of Bollgard and Dipel to S. praeincisus under field conditions where exposition is lower and other food sources besides leaves of Bt plants are available. The method for toxicological tests described here can be adapted to other species of Oribatida, consisting on a new option to risk assessment studies.     

Factors affecting the distribution of a predatory mite on greenhouse sweet pepper

The predatory mite Neoseiulus cucumeris is used for biological control of phytophagous mites and thrips on greenhouse cucumber and sweet pepper. In a previous study, N. cucumeris provided effective control of broad mite but was only rarely found on the sampled leaves, raising questions about the factors affecting N. cucumeris distribution. To determine the distribution of N. cucumeris, leaves of pepper plants were sampled three times per day: just after sunrise, at noon and just before sunset for two years and throughout a 24 h period in one year. The presence of other mites and insects was recorded. Biotic (pollen) and abiotic (temperature, humidity) factors were monitored from the three plant levels. The effect of direct and indirect sunlight on the mites was assessed. N. cucumeris was found primarily in flowers; however, the mite’s distribution was affected by other predators (intraguild predation); in the presence of the predatory bug Orius laevigatus virtually no mites occurred in the flowers. Whereas temperature and humidity varied from the top to the lower level of the plants, apparently neither these factors nor the presence of pollen outside the flowers influenced mite distribution. N. cucumeris was found to be negatively phototropic; therefore N. cucumeris were pre-conditioned to light by rearing under light conditions for 4 months before being released. The light-reared mites were initially more numerous during the noon sampling period, however, rearing conditions caused only a temporary and non-significant change in distribution.     

甘蔗内生解淀粉芽孢杆菌CGB15的分离、鉴定及生防活性

真菌病害占作物病害种类的一半以上,病原真菌是目前已知种类最多的作物病原菌。从作物根际与/或体内分离筛选具有生防活性的微生物,并应用于病害的防控,是除作物品种改良与化学防治外的另一种高效的病害防控策略。【目的】本研究拟筛选并分离鉴定对重要作物病原真菌具有拮抗作用的甘蔗内生细菌,为开发生物防治作物真菌病害新策略提供理论依据。【方法】采用平板对峙法初步筛选对病原真菌具有拮抗能力的甘蔗叶片内生细菌,通过16SrRNA基因测序鉴定其种属;进一步检测候选拮抗内生细菌对甘蔗鞭孢堆黑粉菌(Sporisorium scitamineum)致病发育过程关键步骤：有性配合/菌丝生长、冬孢子萌发的抑制率,田间试验检测其对甘蔗鞭黑穗病的防治效果;检测候选拮抗内生细菌对稻梨孢菌(Pyricularia oryzae)附着胞形成、离体叶片及盆栽条件下叶片病斑形成的抑制作用。【结果】分离自甘蔗叶片的细菌菌株,编号为CGB15,经分子鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。CGB15菌株能有效抑制甘蔗鞭孢堆黑粉菌有性配合/菌丝生长,对峙培养条件下使真菌菌落呈现光滑;抑制冬孢子萌发,...     

Expression of soybean proteinase inhibitors in transgenic sugarcane plants: effects on natural defense against Diatraea saccharalis

The introduction and expression of proteinase inhibitor encoding genes into sugarcane (Saccharum officinarum L.) genome is an interesting strategy for conferring partial resistance to the sugarcane borer Diatraea saccharalis (Lepidoptera: Crambidae), the major insect pest of sugarcane in Brazil. To investigate the role of soybean (Glycine max L.) Kunitz trypsin inhibitor (SKTI) and soybean Bowman–Birk inhibitor (SBBI) in the control of D. saccharalis, the cDNAs encoding these proteinase inhibitors were placed under the control of the maize ubiquitin promoter (Ubi-1), and introduced into sugarcane callus using particle bombardment. Putative transgenic plants were initially identified after regeneration from callus growing in the presence of 30 mg l^–1 geneticin, while molecular characterization of transgenic plants revealed that both genes were incorporated into the sugarcane genome and expressed. We also carried out insect feeding trials using D. saccharalis neonate larvae and leaf tissue excised from propagated transgenic and untransformed plants, and found that the growth of larvae feeding on leaf tissue from transgenic plants containing BBI and Kunitz inhibitors was significantly retarded as compared to larvae fed on leaf tissue from untransformed plants. In greenhouse trials with transgenic sugarcane plants infested with D. saccharalis neonates, we found that these plants still presented the ‘dead heart’ symptom typically observed in susceptible plants in the field, suggesting that the retardation of the growth of D. saccharalis observed in the laboratory-based feeding trials was not sufficient to prevent this type of damage.     

The influence of host plant species on parasitism of pollen beetles (Meligethes spp.) by Phradis morionellus

We investigated the response of the specialist insect predator Oligota kashmirica benefica (Coleoptera: Staphylinidae) to volatiles from lima bean leaves infested with the spider mite Tetranychus urticae (Acari: Tetranychidae), both in a Y-tube olfactometer and in a field in Kyoto, Japan. Adult male and female predators were significantly more attracted to T. urticae-infested leaves than to clean air. Adult male and female predators were not more attracted to uninfested leaves, artificially damaged leaves, or the spider mites and their visible products when compared to clean air. In a field trap experiment, 12 adult predators were caught in three traps containing T. urticae-infested lima bean plants over 13 days, whereas no adult predators were trapped in three traps containing uninfested lima bean plants during the same period. These results showed that O. kashmirica benefica adults responded to herbivore-induced plant volatiles from T. urticae-infested lima bean leaves under both laboratory and field conditions.     

Effects of Bt maize on the herbivore <Emphasis Type="Italic">Spodoptera littoralis</Emphasis> (Lepidoptera: Noctuidae) and the parasitoid <Emphasis Type="Italic">Cotesia marginiventris</Emphasis> (Hymenoptera: Braconidae).

Recent studies have shown that transgenic insect resistant plants can have negative effects on non-target herbivores as well as on beneficial insects. The study of tritrophic interactions gives insight into the complex mechanisms of food webs in the field and can easily be incorporated into a tiered risk assessment framework. We investigated the effects of transgenic maize (Zea mays) expressing insecticidal proteins derived from Bacillus thuringiensis (Bt maize) on Spodoptera littoralis, a non-target herbivore, and on the hymenopteran parasitoid Cotesia marginiventris. In a laboratory study, S. littoralis larvae were reared for their whole lifespan on a mixture of leaves and stems from 2–4-week old Bt maize plants. S. littoralis survival, developmental times and larval weights were significantly affected by Bt maize diet. However, adult moths, which survived development on Bt maize, were the same size as the adults from the control group.C. marginiventris survival, developmental times and cocoon weights were significantly negatively affected if their S. littoralis host larva had been fed Bt maize. ELISA tests confirmed that S. littoralis larvae ingest high amounts of Cry1A(b) toxin while feeding on Bt maize. In S. littoralis pupae and in C. marginiventris cocoon silk, only traces of the toxin could be detected. No toxin was found in S. littoralis and C. marginiventris adults. Thus the toxin is not accumulating in the trophic levels and in fact appears to be excreted. Our results suggest that the effects on C. marginiventris when developing in susceptible S. littoralis larvae are indirect (host mediated). The biological relevance of those results and the significance of this study in risk assessment are discussed.     

Host plant changes produced by the aphid Sipha flava: consequences for aphid feeding behaviour and growth

Induced plant responses may affect the behaviour and growth of the attacking herbivore insect. The aphid Sipha flava (Forbes) produces reddish spots on the infested leaf of its host plant Sorghum halepense (L.). In order to assess the consequences on the aphid of this presumptive induced plant response, we studied the feeding behaviour and growth of S. flava on previously infested and non-infested leaves of S. halepense. Considering that the reddish pigment could play a defensive role, its effect on aphid survival was determined in artificial diets. In addition, changes in the histology of the leaf and the chemical nature of the induced pigment were also studied. Aphids devoted a significantly shorter total time to non-penetration activities in infested than in non-infested leaves. Time before the first phloem ingestion tended to be shorter in infested leaves. The mean relative growth rate of S. flava nymphs was significantly higher on infested than on non-infested leaves. Survival of aphids on diet containing the reddish extract was not significantly different from that on the control diet. Infestation of S. halepense by S. flava produced a reddish coloration in the leaf, which was identified as an anthocyanin by UV-visible spectrometry. Light microscopy showed that only mesophyll cells of previously infested plants presented swelled, dispersed, and heterogeneously stained chloroplasts with a higher accumulation of starch granules, no grana arranged in stacks, and reduction in the amount of inner membranes (thylakoids), relatively to chloroplasts of non-infested leaves. Scanning electron micrographs of leaf surface revealed reduced presence of crystalline epicuticular waxes of epidermal cells in infested leaves as compared to non-infested ones. The main conclusion is that the attack of S. flava to S. halepense leaves induced plant susceptibility where aphid feeding behaviour and growth were both enhanced on previously infested leaves.