Developing a method for laboratory biosafety testing impacts of transgenic plants on non-target insects

A series of laboratory experiments was carried out with the main objective of contributing to the development of biosafety testing methods for transgenic plants or plants produced by other novel technologies. The adult stage of a New Zealand native weevil, Nicaeana cervina (Broun) (Coleoptera: Curculionidae: Entiminae), was used as a test insect as this has become well‐adapted to introduced pasture species, feeding on both ryegrass [Lolium perenne L. (Poaceae)] and white clover [Trifolium repens L. (Fabaceae)]. Ryegrass plants used in experiments were infected with strains of the symbiotic endophytic fungi Neotyphodium lolii (Latch, M.J. Chr. & Samuels) and Epichloe festucae Leuchtm., Schardl & M.R. Siegel and included a transgenic endophyte, which was modified to eliminate production of the insect deterrent, peramine. Nicaeana cervina was exposed to these endophyte‐infected plants along with endophyte‐free ryegrass. Weevils were also exposed to white clover with high or low levels of cyanogens, and to white clover selections bred conventionally to contain high or low levels of crude protein. These bioassays were carried out sometimes over a series of time intervals, and weevil survival, feeding, and reproductive performance variables were measured. Nicaeana cervina survival was generally high, although feeding and oviposition were variable between experiments despite high replication in some cases. A summary of significant differences obtained across all experiments suggested that data on the proportion of weevils feeding and amounts of leaf material consumed were more often significantly different than treatment effects on survival or reproductive performance. A positive relationship between the proportion of weevils feeding and those ovipositing was found, suggesting that a 50% reduction in feeding would result in a 28% reduction in the proportion of weevils ovipositing. Power analysis using the range of values recorded for the variables measured indicated that for proportions of weevils surviving, feeding, or fecund, about 35–42 replicates would be required for a 40% difference in values. For measures of leaf consumption and numbers of eggs per female, fewer replicates were required for 95% power when these values were at the higher end of the range than when they were at the lower end of the observed range. The usefulness of the case study test insect and risk assessment protocols used for biosafety testing are discussed.     

苇状羊茅内生真菌与多年生黑麦草内生真菌实时荧光PCR检测研究

苇状羊茅内生真菌Neotyphodium coenophialum和多年生黑麦草内生真菌N．lolii对美国、新西兰等国家的畜牧业曾经造成过巨大损失。以N．coenophialum和N．lolii及其近似种N．huerfanum、N．chisosum、N．aotearoae、N．sp．共6种18个菌株,以及苇状羊茅和多年生黑麦草8个品种种子为供试材料,根据Tub-2基因设计了通用Taqman探针及引物,根据NC25基因设计了N．coenophialum和N．lolii的特异Taqman探针及共用引物,通过通用探针的单色荧光PCR和特异探针的双色荧光PCR,建立了N．coenophialum和N．lolii的菌丝及单粒种子稳定可靠、特异性强的荧光PCR检测方法,检测灵敏度达到单粒种子,使检测时间由至少一个月缩短至7～8个小时。     

Fungal endophyte infection increases tall fescue's survival,growth, and flowering in a reconstructed prairie

Grasslands in North America are increasingly threatened by land conversion and ecological degradation, prompting restoration efforts to increase native plant species diversity and improve wildlife habitat. A major challenge is the removal and management of nonnative invasive species such as tall fescue (Schedonorus arundinaceus), which has a symbiotic association with a fungal endophyte (Epichloë coenophiala) that modifies its ecological interactions. Using transplanted clumps of the cultivar Kentucky‐31, we tested the effects of endophyte infection on tall fescue's survival and performance (tiller production, flowering, and basal area) for 5 years in a central Kentucky reconstructed prairie. We predicted that endophyte infected (E+) clumps would have increased performance compared to endophyte‐free (E?) clumps. Overall, E+ clumps had greater survival, tiller production, flowering tiller production, and basal area, but not reproductive effort (proportion of tillers flowering) as compared to E? clumps. However, survival and trends in tiller number and basal area over the 5‐year period suggested experimental tall fescue populations were in decline in the reconstructed prairie, although the E? population declined more rapidly. Our study provides evidence that endophyte infection improved tall fescue's growth and survival in a postreconstruction plant community, at least in the early years following reconstruction, and may increase the invasive potential of this nonnative species in prairie restorations.     

Genetic diversity in epichloid endophytes of Hordelymus europaeus suggests repeated host jumps and interspecific hybridizations

Epichloid fungal endophytes (Epichloë and Neotyphodium spp.) are excellent model systems for studying speciation processes because of their variable life history traits that are linked to host grass fitness. Presumed jumps to new hosts and subsequent somatic hybridizations appear to be common among epichloid endophytes resulting in increased genetic variation upon which selection can act and speciation be initiated. In this study, we explored the endophyte diversity of a rare European native woodland grass species, Hordelymus europaeus, along a latitudinal transect covering the entire distribution range of H. europaeus. From 28 populations in six countries, isolates were sampled and molecularly characterized. Based on the sequences of tubB and tefA, six distinct epichloid taxa (interspecific hybrid or cryptic haploid species) were found, of which four were novel and two have been previously reported from this host. Of the novel endophytes, two were presumed to be interspecific hybrids and two of nonhybrid origin. While previously known endophytes of H. europaeus are seed‐born and strictly asexual, one of the novel nonhybrid endophytes found in the glacial refugium of the Apennine peninsula reproduced sexually in cultured plants. This is the first case of a seed‐borne, but sexually reproducing endophyte of this host. We discuss the origin, and possible ancestral species, of the six epichloid taxa using phylogenetic analyses. Repeated host jumps and somatic hybridizations characterize the diversity of the endophytes. To date, no other grass species is known to host a larger diversity of endophytes than H. europaeus.     

内生真菌感染对3个不同地理种群羽茅光合特性的影响

研究了内蒙古中东部草原3个不同样地（羊草、西乌旗和霍林河）中Neotyphodium内生真菌的侵染对宿主羽茅(Achnatherum sibiricum(L.) Keng)种群光合生理特性的影响,结果表明：自然条件下,羊草样地和西乌旗样地染菌(E+)羽茅植株的净光合速率(P_n)显著高于不染菌(E-)羽茅,说明E+羽茅植株具有更高的同化能力,而霍林河样地中二者差异不显著;3个样地中Neotyphodium内生真菌对宿主羽茅的蒸腾速率、气孔导度、光能利用效率、水分利用效率均无显著影响;引起羽茅植株午间P_n降低的主要原因是非气孔因素,与是否染菌没有必然联系;光响应曲线拟合结果表明Neotyphodium内生真菌对宿主羽茅的表观量子效率、最大净光合速率、暗呼吸速率、光补偿点和光饱和点没有显著影响。     

Effects of endophytic fungi on some drought tolerance mechanisms of tall fescue in a hydroponics culture

Neotyphodium, a seed-transmissible nonpathogenic fungal endophyte (symbiont) is considered beneficial because endophyte-infected grasses are more drought-tolerant, produce more dry matter, utilize soil nitrogen more efficiently, and deter insects. In this study, the effects of endophytes on physiological mechanisms of drought tolerance in tall fescue (Festuca arundinacea Schreb.) were studied in a greenhouse. Two clonally propagated genotypes of tall fescue (F. arundinacea Schreb.), naturally containing endophyte (EI), and their endophyte-free ramets (EF) were tested at three water stress treatments exerted by PEG 6000 in a hydroponics system. Relative water content (RWC), cell membrane stability (CMS), proline and chlorophyll contents in plant leaves were measured during water stress treatments. After harvest, K⁺, Ca²⁺, and Mg²⁺ contents were measured in plant roots and shoots. After 20 days under stress conditions, plants were transferred to basal hydroponics medium, and their survival after stress relief was evaluated. The results showed that endophyte considerably contributes to host grass water stress tolerance. Both genotypes of EI and EF plants did not differ in RWC, but, regardless of the infection status, genotype 75 had the higher RWC than genotype 83. EI clones of both genotypes maintained slightly higher chlorophyll content and membrane stability than EF clones, although these differences were not significant. The EI plants of genotype 83 concentrated significantly more proline than EF plants, but in the genotype 75, differences between EI and EF clones were not significant. Plant mineral absorption was also influenced by the endophyte presence. EI clones had the higher concentrations of K⁺ in the shoots of both genotypes. The Mg²⁺ and Ca²⁺ contents in EF plants of both genotypes were higher than EI plants in the roots, but in the shoots there were no differences between EI and EF clones. EI clones survived longer after stress removal. These results strongly suggest that Neotyphodium endophytes exert their effects on tall fescue drought tolerance through alteration of various physiological mechanisms involved. Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 4, pp. 563–570. This test was submitted by the authors in English.     

Influence of temperature on alkaloid levels and fall armyworm performance in endophytic tall fescue and perennial ryegrass

The symbiotic relationships between Neotyphodium endophytes (Clavicipitacea) and certain cool‐season (C3) grasses result in the synthesis of several alkaloids that defend the plant against herbivory. Over a 3 month period we evaluated the effects of temperature on the expression of these alkaloids in tall fescue, Festuca arundinacea Schreb, and perennial ryegrass, Lolium perenne L. (Poaceae). Response surface regression analysis indicated that month, temperature, and their interaction had an impact on the alkaloid levels in both grasses. We aimed to identify the alkaloids most closely associated with enhanced resistance to the fall armyworm, Spodoptera frugiperda JE Smith (Lepidoptera: Noctuidae), and clarify the role of temperature in governing the expression of these alkaloids. The dry weights and survival of fall armyworms feeding on endophyte‐infected tall fescue or perennial ryegrass were significantly lower than for those feeding on uninfected grass, whereas endophyte infection had no significant influence on survival. For tall fescue, a four‐alkaloid model consisting of a plant alkaloid, perloline, and the fungal alkaloids ergonovine chanoclavine, and ergocryptine, explained 47% of the variation in fall armyworm dry weight, whereas a three‐alkaloid model consisting of the plant alkaloid perloline methyl ether and the fungal alkaloids ergonovine and ergocryptine explained 70% of the variation in fall armyworm dry weight on perennial ryegrass. Although temperature had a significant influence on overall alkaloid expression in both grasses, the influence of temperature on individual alkaloids varied over time. The levels of those alkaloids most closely linked to armyworm performance increased linearly or curvilinearly with increasing temperature during the last 2 months of the study. We conclude that the growth temperature of grasses can influence the performance of fall armyworm, and that this effect may be mediated through a set of plant‐ and endophyte‐related alkaloids.     

Endophytic fungi alter relationships between diversity and ecosystem properties

Recent studies have expanded research on biodiversity by investigating whether the effects of diversity on ecosystem functioning hinge on the presence of symbiotic microorganisms. Cool‐season grasses commonly harbour endophytic fungi that can enhance plant resistance to herbivory, drought and competition. We address whether these endosymbionts modify relationships between diversity and two ecosystem properties: productivity and invasibility. We develop a graphical model that predicts endophyte infection of a grass host will weaken correlations between diversity and ecosystem properties. We then use a long‐term field experiment to test this prediction by manipulating symbiosis in tall fescue grass (Festuca arundinacea), a common and invasive species in the US. As predicted, endophyte infection reduced the strength of correlations between diversity and both primary productivity and the invasiveness of tall fescue. By altering relationships between diversity and ecosystem functioning, endophytic fungi may contribute more to the dynamics of communities than previously supposed.     

Gas Exchange and Chlorophyll Fluorescence in Symbiotic and Non-Symbiotic Ryegrass Under Water Stress

The symbiotic association of endophyte fungus, Neotyphodium lolii, and ryegrass improves the ryegrass resistance to drought. This is shown by a 30 % increase in the number of suckers in infected plants (E+), compared to plants lacking endophyte (E−), and by a higher water potential in the E+ than E− plants. The E+ plants have higher stomatal conductance (g _s), transpiration rate, net photosynthetic rate (P _N), and photorespiratory electron transport rate than the E− plants. The maximal photochemical efficiency (F_v/F_m) and the actual photochemical efficiency (Φ_PS2) are not affected by the endophyte fungus. The increase in P _N of the E+ plants subjected to water stress was independent from internal CO₂ concentration. An increased P _N was observed in E+ plants also in optimal water supply. Hence the drought resistance of E+ plants results in increased g _s, P _N, and photorespiratory electron transport rate. This revised version was published online in June 2006 with corrections to the Cover Date.