Origin of a sudden mass occurrence of the stolbur phytoplasma vector Hyalesthes obsoletus (Cixiidae) in Austria

The grapevine disease ‘bois noir’ is widespread in European viticulture, but in many regions there is a lack of correspondence between disease spread and abundance of the main insect vector, the planthopper Hyalesthes obsoletus. This was the situation in Austria until 2012, when a mass occurrence of the vector was observed on Urtica dioica, a new host plant for the vector and reservoir plant for the pathogen, stolbur phytoplasma, in this area. Here we analyse the origin of the Austrian vector populations using genetic markers. The origin was unambiguously assigned to two regional populations, and two causes for the population expansion: immigration of East Central European populations and local demographic expansion. The observed population increase was thus independent of phylogenetic ancestry, but linked to the host plant and the exchange of a specific stolbur phytoplasma strain between the two vector populations. These circumstances are identical to but independent of the emergence of bois noir west of the European Alps, where an exchange between other vector populations associated with U. dioica of another stolbur phytoplasma genotype has led to disease outbreaks. Combined, the independent outbreaks in Austria and Europe west of the Alps are suggestive of an active role for stolbur phytoplasma in the vector–plant interaction and thus the host distribution of the vector.     

Host plant preferences of Hyalesthes obsoletus,the vector of the grapevine yellows disease ‘bois noir’, in Switzerland

Bois noir is an important grapevine yellows disease in Europe that can cause serious economic losses in grapevine production. It is caused by stolbur phytoplasma strains of the taxonomic group 16Sr‐XII‐A. Hyalesthes obsoletus Signoret (Hemiptera: Cixiidae) is the most important vector of bois noir in Europe. This polyphagous planthopper is assumed to mainly use stinging nettle [Urtica dioica L. (Urticaceae)] and field bindweed [Convolvulus arvensis L. (Convolvulaceae)] as its host plants. For a better understanding of the epidemiology of bois noir in Switzerland, host plant preferences of H. obsoletus were studied in the field and in the laboratory. In vineyards of Western Switzerland, adults of H. obsoletus were primarily captured on U. dioica, but a few specimens were also caught on C. arvensis, hedge bindweed [Calystegia sepium (L.) R. Brown (Convolvulaceae)], and five other dicotyledons [i.e., Clematis vitalba L. (Ranunculaceae), Lepidium draba L. (Brassicaceae), Plantago lanceolata L. (Plantaginaceae), Polygonum aviculare L. (Polygonaceae), and Taraxacum officinale Weber (Asteraceae)]. The preference of the vector for U. dioica compared to C. arvensis was confirmed by a second, more targeted field study and by the positioning of emergence traps above the two plant species. Two‐choice experiments in the laboratory showed that H. obsoletus adults originating from U. dioica preferred to feed and to oviposit on U. dioica compared to C. arvensis. However, H. obsoletus nymphs showed no host plant preference, even though they developed much better on U. dioica than on C. arvensis. Similarly, adults survived significantly longer on U. dioica than on C. arvensis or any other plant species tested [i.e., L. draba and Lavandula angustifolia Mill. (Lamiaceae)]. In conclusion, although nymphs of H. obsoletus had no inherent host plant preference, adults tested preferred to feed and oviposit on U. dioica, which is in agreement with the observed superior performance of both nymphal and adult stages on this plant species. Urtica dioica appears to be the principal host plant of H. obsoletus in Switzerland and plays therefore an important role in the epidemiology of the bois noir disease in Swiss vineyards.     

Sympatric diversification vs. immigration: deciphering host‐plant specialization in a polyphagous insect,the stolbur phytoplasma vector Hyalesthes obsoletus (Cixiidae)

The epidemiology of vector transmitted plant diseases is highly influenced by dispersal and the host‐plant range of the vector. Widening the vector's host range may increase transmission potential, whereas specialization may induce specific disease cycles. The process leading to a vector's host shift and its epidemiological outcome is therefore embedded in the frameworks of sympatric evolution vs. immigration of preadapted populations. In this study, we analyse whether a host shift of the stolbur phytoplasma vector, Hyalesthes obsoletus from field bindweed to stinging nettle in its northern distribution range evolved sympatrically or by immigration. The exploitation of stinging nettle has led to outbreaks of the grapevine disease bois noir caused by a stinging nettle‐specific phytoplasma strain. Microsatellite data from populations from northern and ancestral ranges provide strong evidence for sympatric host‐race evolution in the northern range: Host‐plant associated populations were significantly differentiated among syntopic sites (0.054 < F_HT < 0.098) and constant over 5 years. While gene flow was asymmetric from the old into the predicted new host race, which had significantly reduced genetic diversity, the genetic identity between syntopic host‐race populations in the northern range was higher than between these populations and syntopic populations in ancestral ranges, where there was no evidence for genetic host races. Although immigration was detected in the northern field bindweed population, it cannot explain host‐race diversification but suggests the introduction of a stinging nettle‐specific phytoplasma strain by plant‐unspecific vectors. The evolution of host races in the northern range has led to specific vector‐based bois noir disease cycles.     

Controlling ‘bois noir’ disease on grapevine: does the timing of herbicide application affect vector emergence?

Bois noir is an important grapevine yellows disease that can cause serious economical losses in European grapevine production. Hyalesthes obsoletus Signoret (Hemiptera, Cixiidae) is the principal vector of bois noir in Switzerland and stinging nettle (Urtica dioica) is its favourite host plant species in vineyards. As bois noir disease can hardly be cured and direct control measures against H. obsoletus are ineffective, viticultural control practices target stinging nettle, the actual reservoir and source of both the pathogen and its vector. Currently, it is recommended to apply herbicides against stinging nettle at the end of the season to kill developing H. obsoletus nymphs. To verify if this late period of herbicide application is justified, stinging nettle patches were treated with glyphosate in the autumn, in the spring or were left untreated as a control. Herbicide applications at both dates controlled the growth of stinging nettle very well in the subsequent summer, although the autumnal treatment was slightly more efficient. To study glyphosate’s direct impact on the development of H. obsoletus nymphs, emergence traps were placed directly in the centre of treated and untreated stinging nettle patches. There was no significant difference among the three treatments in the total number of adults emerging. Thus, an aerial application of glyphosate in either spring or autumn did not inhibit the nymphs’ development on the roots of stinging nettle in the soil. Our results challenge current recommendations of applying herbicides against stinging nettle at the end of the season and suggest that stinging nettle could also be controlled in spring, alike other viticultural weeds.     

Invasion biology and host specificity of the grapevine yellows disease vector Hyalesthes obsoletus in Europe

Within the past 10 years, the yellows disease ‘bois noir’ (BN) has become one of the commercially most important diseases of grapevine [Vitis vinifera L. (Vitaceae)] in Europe. Infection pressure is caused by phytoplasmas of the stolbur 16SrXII‐A group that are transmitted by a planthopper vector, Hyalesthes obsoletus Signoret (Homoptera: Auchenorrhyncha). Infestation happens as an accidental side‐effect of the feeding behaviour of the vector, as vector and pathogen proliferation is dependent on other plants. In Germany, the increase of BN is correlated with the use of a new host plant by the vector, increase in abundance of the vector on the new host plant, and dissemination of host plant‐specific pathogen strains. In this article, we investigate geographic and host‐associated range expansion of the vector. We test whether host‐plant utilization in Germany, hence the increase in BN, is related to genetic host races of the vector and, if so, whether these have evolved locally or have immigrated from southern populations that traditionally use the new host plant. The genetic population analysis demonstrates a recent expansion and circum‐alpine invasion of H. obsoletus into German and northern French wine‐growing regions, which coincides with the emergence of BN. No H. obsoletus mitochondrial DNA haplotype host‐plant affiliation was found, implying that the ability to use alternative host plants is genetically intrinsic to H. obsoletus. However, subtle yet significant random amplified polymorphic DNA (RAPD) genetic differentiation was found among host plant populations. When combined, these results suggest that a geographic range expansion of H. obsoletus only partly explains the increase of BN, and that interactions with host plants also occur. Further possible beneficial factors to H. obsoletus, such as temperature increase and phytoplasma interactions, are discussed.     

Phytoplasma Transmission by Heterologous Grafting Influences Viability of the Scion and Results in Early Symptom Development in Periwinkle Rootstock

The stolbur phytoplasma ‘Candidatus Phytoplasma solani’ is responsible for the grapevine disease ‘bois noir’ affecting a number of wine‐growing areas in Europe. Transmission of stolbur phytoplasma to different laboratory hosts can be difficult due to the requirement of transmitting insect vectors or parasite plants. Here, heterologous grafting was used as an alternative technique for transmission of common and strongly symptomatic stolbur genotypes CPsM4_At1 and CPsM4_At6 of ‘Ca. P. solani’ to experimental host plants such as Catharanthus roseus and tomato making phytoplasma strains more accessible for molecular and experimental investigations in different plant species. Transmission was confirmed by quantitative PCR, microscopy and nested PCR followed by marker gene sequencing. In our study, the transmission of different genotypes of ‘Ca. P. solani’ resulted in distinguishable symptom development in the laboratory host C. roseus. Symptom development in grafted rootstock was observed three to 7 weeks after heterologous grafting. Survival of the graft unit was influenced by the presence of ‘Ca. P. solani’ in the scions and was clearly reduced in phytoplasma free scion – rootstock combinations.     

Infection of Bois‐Noir tuf‐type‐I stolbur phytoplasma in Hyalesthes obsoletus (Hemiptera: Cixiidae) larvae and influence on larval size

Recent dramatic spread of the grapevine yellows disease Bois Noir (BN) in Germany is above all explained by highly increased abundances of the vector Hyalesthes obsoletus (Hemiptera: Cixiidae) associated to the plant Urtica dioica, the reservoir of the BN pathogen stolbur tuf‐type‐I. The vector acquires BN‐phytoplasma as larvae whilst feeding on the roots of infected U. dioica. To understand the dynamics of the Urtica‐cycle, we tested at what instar larvae become infected and whether infection affects larvae size (i.e. growth) at two sites in the Mosel Valley, Germany. Larvae were tested from infected plants and collected at instar‐stages 3, 4 and 5. Larvae at stage 3 were already infected but infection rates increased significantly between stage 3 and 5, mean infection rates: 0.12–0.62. There was no effect of infection on larval size at any instar stage.     

In vitro alpha-amylase inhibition and in vivo antioxidant potential of Momordica dioica seeds in streptozotocin-induced oxidative stress in diabetic rats

Momordica dioica Roxb. Commonly known as “Kakora” in Telugu, is used in the Indian traditional system of medicine for the treatment of diabetes. The aim of this study was to investigate the antidiabetic activity of methanolic extract of M. dioica seeds (MEMD) in streptozotocin (STZ) induced diabetic rats. The in vitro α-amylase inhibitory activity of the MEMD was done by spectrophotometric method. Diabetes was induced by STZ (45 mg/kg; i.p), MEMD (100 & 200 mg/kg; b.wt) and standard drug metformin (50 mg/kg; b.wt) were administered to the diabetic rats. Blood glucose was estimated on the 11th day and the level of MDA, SOD and CAT was estimated in the liver tissue homogenate after the 15 days of experimental period. MEMD showed significant inhibition of alpha amylase activity and the IC₅₀ was found to be 48 μg/ml. Oral administration of MEMD significantly reduced blood glucose level (P < 0.05), diminished the MDA level and refurbished depleted antioxidant enzymes and Insulin level to normalcy. These findings revealed that M. dioica seeds possess antihyperglycemic, antioxidant and anti lipid peroxidative activity and thus mitigate STZ-induced oxidative damage.     

Screening for hydroxynitrile lyase activity in non-commercialised plants

Hydroxynitrile lyases are used for the synthesis of enantiomerically pure cyanohydrins which are of great importance in the pharmaceutical and fine chemical industries. In this study, the hydroxynitrile lyase activity of 100 plants from 40 families was investigated, first by screening for cyanogenic activity, followed by a hydroxynitrile lyase activity assay. Of the 100 plants, four were found to be cyanogenic and exhibited specific hydroxynitrile lyase activity: Adenia sp. (0.44 U/mg), Adenia firingalavensis (2.88 U/mg), Adenia fruticosa (1.99 U/mg) and, Adenia pechuelii (2.35 U/mg), all from the family Passifloraceae. This is the first report of hydroxynitrile lyase activity in these plants.     

Evaluating the potential of buckwheat and cahaba vetch as nectar producing cover crops for enhancing biological control of Homalodisca vitripennis in California vineyards

The glassy-winged sharpshooter, Homalodisca vitripennis (Germar) is a significant pest of grapevines in California due to vectoring Xylella fastidiosa Wells et al., Buckwheat (Fagopyrum esculentum Moench) and vetch (Vicia sativa L. cv. ‘cahaba white’) are potential nectar-producing cover crop plants in California vineyards that could enhance Gonatocerus ashmeadi Girault, a parasitoid of H. vitripennis. Three aspects of these 2 plant species were evaluated. (1) Effect on parasitoid fitness: In the laboratory, buckwheat and vetch enhanced survival of G. ashmeadi by 9 and 6 days, respectively, compared to water. G. ashmeadi offspring production was 81% and 142% greater when females were provided vetch or buckwheat, respectively, compared to water. Buckwheat decreased G. ashmeadi female offspring by 19% compared to water and vetch. (2) Nectar production phenology in southern California: From April through September, buckwheat required only 23–32 days from sowing to nectar-producing flowers. Vetch required an additional 14–33 days to produce extrafloral nectar during June–August. The range in length of the nectar producing period between August 2007 and November 2007 was 41–52 days and 163–164 days for buckwheat and vetch, respectively. (3) X. fastidiosa Wells et al. host testing and H. vitripennis transmission studies: Needle inoculation of cover crop plants showed that X. fastidiosa successfully infected and replicated in buckwheat, vetch, sweet alyssum [Lobularia maritima (L.) Desv.] and coriander (Coriandrum sativum L.), plants that were assessed here or elsewhere as nectar producing cover crops for use in vineyards. H. vitripennis successfully transmitted X. fastidiosa between buckwheat and grapevine in the greenhouse and field. H. vitripennis successfully transmitted X. fastidiosa from grapevine to vetch in the field, while transmission studies investigating movement from vetch to grapevine in the greenhouse were inconclusive.     

Population structure and incidence of the stolbur phytoplasma vector Hyalesthes obsoletus (Cixiidae) among geographic regions in Switzerland

The dissemination of stolbur phytoplasma (16Sr‐XIIA group)‐induced yellows diseases depends on the dispersal biology and host plant fidelity of the planthopper vector Hyalesthes obsoletus (Hemiptera: Cixiidae). We analysed the degree of these two properties in H. obsoletus by studying its population genetic structure and stolbur infection rates relative to the two major host plants, Convolvolus arvensis and Urtica dioica, in order to infer relevant divisions for stolbur epidemiology in Swiss viticultural regions. Three regional populations with the potential to determine stolbur epidemiology in distinct ways were identified. First, populations associated with U. dioica in northern Switzerland were most related to genetically distinct U. dioica host race populations identified previously in Germany. Second, populations in central and southwest Switzerland were undifferentiated relative to host plant and likely have wider stolbur transmission breadths than the northern specialized populations. Third, populations in south of the Alps (Ticino) were undifferentiated relative to host plant but geographically isolated from other Swiss regions, thus implying separate population dynamics in this area. The knowledge of these three distinct epidemiological cycles will help to adapt management programmes against stolbur diseases in Swiss vineyards.     

Field assessment,in Greece and Russia,of the facultative saprophytic fungus,Colletotrichum salsolae,for biological control of Russian thistle (Salsola tragus)

Russian thistle (Salsola tragus, tumbleweed, RT) is a problematic invasive weed in the United States (U.S.) and is a target of biological control efforts. The facultative saprophytic fungus Colletotrichum salsolae (CS) kills RT plants in greenhouse tests and is specific to Salsola spp., which are not native in the U.S. However, the effectiveness of CS in controlling RT has not been previously demonstrated. The objectives of this study were to determine in field tests: (1) disease progress of CS in time; (2) the relationship of disease progress to rainfall and temperature; (3) the effect of CS on RT plant density. Field tests were established in Serres and Kozani, Greece and Taman and Tuzla, Russia with isolates of the pathogen collected in the respective countries. Solid inoculum was prepared by asceptically inoculating sterile mixtures of grain and grain hulls with axenic cultures of CS. Spore suspensions used in Russia were prepared by blending pure sporulating cultures of CS with distilled water and diluting the suspension to 10⁶ conidia per ml. Six field plots, each subdivided into 36 subplots, of an RT infested field in Serres, Greece were inoculated on October 23, 2006 by placing about 300 g of solid inoculum in the center of each plot. Four field plots, similarly subdivided, in each of two fields at Kozani, Greece were inoculated in the same way on October 1, 2010. RT density was counted and recorded in each sub-plot prior to inoculation and in September in each of 2 years following inoculation. Disease incidence and/or severity in each sub-plot were recorded at about 7-days intervals after inoculation. Rainfall and temperature data, from inoculation until 40–55 days after inoculation, were collected and recorded at Serres and at the Kozani airport meteorological station. Disease progressed rapidly at both sites and was correlated with cumulative rainfall. By 2 years after inoculation, RT had been eliminated from the Serres site and one field in Kozani. In the other Kozani field, RT density declined to 0–25% from original densities of about 80% in large areas of the field. RT plants in Taman and Tuzla, Russia were inoculated either with 250 g of grain inoculum or with a suspension of 10⁶ conidia sprayed onto each plant until runoff. The proportion of diseased tissue reached 1.0 by 55 days after inoculation in both sites. Non-inoculated plants that were near inoculated plants became diseased quickly and reached the same disease severity as inoculated plants. Disease severity was correlated with cumulative rainfall but not temperature. This pathogen and inoculation procedure offers a low-cost solution to RT infestations. Since CS is specific to Salsola spp., this effective biological control is also environmentally friendly.     

First detection and molecular characterization of grapevine phytoplasmas in Kosovo

A search for phytoplasma-associated diseases was conducted for the first time in the main grapevine-growing localities of the Dukagjini plain in Kosovo. A total of 144 samples were collected from grapevine cultivars displaying leaf yellowing, reddening, discolouration and irregular wood ripening, and analysed using nested and quantitative PCR assays. These assays showed that 35.4% of samples belonging to eight cultivars were positive to the presence of phytoplasmas in the 16SrXII group. The 16S rDNA phytoplasma sequences obtained from 15 samples shared identity greater than 99.5% with ‘Candidatus Phytoplasma solani’. Sequence analysis of the tuf gene showed that the strains found in Kosovar grapevines are in the tuf-type b1 group, sharing 99.6% to 99.8% identity with ‘Ca. P. solani'-related strains associated with the “bois noir” grapevine disease in many European countries; the secY gene sequences, on the other hand, shared 100% identity with ‘Ca. P. solani' strains from Bosnia and Herzegovina, Serbia, Croatia and Turkey. This study constitutes the first report on the presence and molecular characterization of phytoplasmas in Kosovar vineyards. Based on these results, it is recommended that testing for phytoplasma be included in the certification program for grapevine in Kosovo.     

Vitex agnus‐castus cannot be used as trap plant for the vector Hyalesthes obsoletus to prevent infections by ‘Candidatus Phytoplasma solani’ in northern Italian vineyards: Experimental evidence

Bois noir (BN), the most prevalent disease of the grapevine yellows complex, causes considerable yield loss in vineyards. BN is associated with phytoplasma strains of the species ‘Candidatus Phytoplasma solani’ (taxonomic subgroup 16SrXII‐A). In Europe, the BN phytoplasma is transmitted to grapevine mainly by Hyalesthes obsoletus, a polyphagous cixiid completing its life cycle on stinging nettle and field bindweed. As a result of the complexity of BN epidemiology, no effective control strategies have been developed. In previous studies conducted in the eastern Mediterranean coast of Israel, chaste tree (Vitex agnus‐castus) was found to be the preferred host plant of H. obsoletus but did not harbour BN phytoplasma. Thus, a ‘push and pull’ strategy was suggested based on the fact that chaste tree plants located at vineyard borders was an effective trap plant for H. obsoletus adults. However, in other studies carried out in the eastern Adriatic coast of Montenegro, chaste tree was found to be a key source plant for BN phytoplasma transmission to grapevine. This study aimed to investigate (i) the interaction between chaste tree and H. obsoletus through survival, attractiveness and oviposition experiments conducted comparing the behaviour of H. obsoletus in chaste tree versus stinging nettle and grapevine and (ii) the capability of chaste tree to harbor ‘Ca. P. solani’ in northern Italy through transmission trials. H. obsoletus adults were found to survive on chaste tree and grapevine over a 1 week period and prefer chaste tree to grapevine. Moreover, H. obsoletus produced eggs and overwintered as nymphs on chaste tree, even if at a lesser extent than on stinging nettle. H. obsoletus originating from nettle was found able to transmit ‘Ca. P. solani’ to chaste tree (2 plants of 16 were found infected by the BN phytoplasma strain St5 identified in H. obsoletus specimens). These results increased our knowledge about the role of Vitex agnus‐castus as host plant of H. obsoletus and BN phytoplasma in northern Italy and do not recommend considering chaste tree as trap plant at vineyard borders.     

Characterization and early detection of grapevine (Vitis vinifera) stress responses to esca disease by in situ chlorophyll fluorescence and comparison with drought stress

Esca disease, as well as other trunk diseases of grapevine, is an important wood disease that impedes the water transport in plants by clogging the xylem vessels. This type of effect is not detectable for years, due to the long latency time of the disease. In a field experiment, the susceptibility of Vitis vinifera cv. Cabernet sauvignon and Merlot to esca disease was evaluated by visual assessment of foliar symptoms and by necrosis and white rot indexes. C. sauvignon was highly susceptible and Merlot was tolerant to esca. The characteristics of fast chlorophyll a fluorescence transient were investigated in attached leaves by using the so-called JIP-test. The fluorescence transient was analyzed and plants without visible esca foliar symptoms were compared with those showing symptoms. In C. sauvignon, alteration of the photosynthetic apparatus could be detected 2 months before the appearance of foliar symptoms in autumn. To our knowledge, this is the first report of early detection of esca disease using a nondestructive method. For Merlot, only one JIP-test parameter was affected. However, when both cultivars were compared, the relationship of the performance index (defined by the density of reaction centers (RCs) and by the yields φ_Po and ψ_o; PI_ABS = [RC/ABS][φ_Po/(1 − φ_Po)][ψ_o/(1 − ψ_o)]) versus the calculated rate of the electron transfer [probability that an absorbed photon moves an electron further than $Q_{\text{A}}^{-}$; φ_Eo = φ_Poψ_o = (TR_o/ABS)(ET_o/TR_o) = ET_o/ABS = (1 − F₀/F_M)(1 − V_J)] permitted us to separate the highest performing cultivar Merlot from the susceptible C. sauvignon. Also, the method used allowed us to detect modification of the photosystem II (PS II) performance in greenhouse-grown Riesling × Sylvaner after a drought stress. Finally, the comparison of the fluorescence transients of esca-affected and drought-stressed grapevines provided information on the differentiated functional-behavior patterns of PS II for the two stress types. These results suggest that esca infection cannot simply be interpreted as a water transport deficit through xylem dysfunction, but that other reaction mechanisms in the plants must be considered. The possibility to use fast chlorophyll a fluorescence monitoring as a wood decay early detection tool is discussed.     

Timing of onset of evening activity of adult Chinese rose beetles (Coleoptera: Scarabaeidae)

Adult Chinese rose beetles, Adoretus sinicus (Burmeister) (Coleoptera: Scarabaeidae: Adoretini), present in China, Taiwan, Indonesia, Cambodia, Laos, Singapore, Thailand, Vietnam, the Marianas Islands, the Caroline Islands, and the Hawaiian Islands, are nighttime defoliators that feed on a wide variety of plant species. It has recently been demonstrated that illumination of plants at dusk has the potential to discourage feeding by adult Chinese rose beetles on the illuminated plants. To effectively use lighting to minimize defoliation of host plants, it is critical to know the timing of the initial host plant colonization by the beetles to ensure that illumination is initiated before host plant colonization begins. Adult Chinese rose beetles were observed to colonize host plants at dusk, with initiation of beetle colonization averaging more than 21 min after sunset, with the earliest observed beetle colonization occurring 11 min after sunset. These times corresponded to an average light level of 7.0 lux at the first colonization and the earliest first colonization occurring at 26.9 lux. Based on these results, use of lighting to minimize defoliation of host plants should be initiated at about sunset in order to discourage colonization (and associated defoliation) by adult Chinese rose beetles.     

Effect of infection by Metarhizium anisopliae (Hypocreales: Clavicipitaceae) on the feeding and oviposition of the pea leafminer Liriomyza huidobrensis (Diptera: Agromyzidae) on different host plants

The effect of fungal infection by Metarhizium anisopliae on feeding and oviposition of adult Liriomyza huidobrensis was examined on three host plants, faba bean (Vicia faba), French bean (Phaseolus vuklgaris) and snow pea (Pisum sativum) in the laboratory. Flies were contaminated with dry conidia and allowed to feed and oviposit on the different host plants. Mortality in L. huidobrensis varied between 14% and 20% in the controls and between 77% and 100% in fungal treatments 120 h post-infection for the three host plants. L. huidobrensis made more punctures (47.3–52.6 cm^?2) in the control than in the fungal treatments (23.1–26.9 cm^?2) for the three host plants. The cumulative average number of punctures cm^?2/female by L. huidobrensis was higher in the controls than in fungal treatments from 72 h post-treatment in faba bean (12.2 vs. 8.2) and French bean (14.8 vs. 8.9), and from 48 h post-inoculation in snow pea (8.5 vs. 5.7). Female L. huidobrensis laid more eggs in the control (0.6–6.1) than in fungal treatments (0.2–1.5) across the host plants tested. The cumulative mean number of eggs cm^?2/female was significantly higher in the controls than in fungal treatments from 48 h post-treatment in faba bean (0.4 vs. 0.2) and French bean (0.1 vs. 0), and 96 h post-inoculation in snow pea (0.2 vs. 0.1). The host plant did not affect the average total number of punctures but had a significant effect on egg laying, with faba bean harboring greater number of eggs in both control and fungal treatments. A proper timeline application of the fungus before onset of feeding and oviposition peaks will be crucial in field suppression of the pest using M. anisopliae. In addition, a great consideration must be given to the target host plants prior to application of the fungus.     

Foraging efficiency of Lysiphlebus fabarum Marshall (Hymenoptera: Aphidiidae) parasitizing the black bean aphid,Aphis fabae Scopoli (Hemiptera: Aphididae), under laboratory conditions

Foraging efficiency of Lysiphlebus fabarum Marshall, the most abundant and effective parasitoid of the black bean aphid, Aphis fabae Scopoli, in terms of functional and numerical responses and mutual interference, was studied on broad bean plants in a growth chamber at 21 °C, 70 ± 5% RH and a photoperiod of 16:8 (L:D) h. Using logistic regression, a type II functional response was determined for L. fabarum. Nonlinear least-square regression was used to estimate the searching efficiency (a') and handling time (T_h). Numerical response of L. fabarum to cumulative host densities was partially linearly increasing. Nicholson's model and linear regression were used to determine the per capita searching efficiency and interference coefficient, respectively. In general, the per capita parasitism and, consequently, the per capita searching efficiency decreased significantly as parasitoid density increased. Mutual interference and quest constants were ? 0.508 and ? 0.697, respectively. The results of this study suggest that both the host and parasitoid densities have no significant impact on the progeny sex ratio. The information provided here will be useful in assessing the efficiency of L. fabarum as a biological control agent of A. fabae, devising mass-rearing protocols and implementing release programs for this parasitoid.     

Expression of a bifunctional cellulase with exoglucanase and endoglucanase activities to enhance the hydrolysis ability of cellulase from a marine Aspergillus niger

Low exoglucanase and endoglucanase activities of marine Aspergillus niger cellulase decreased the hydrolyzing ability of cellulase. To increase the activity of halostable cellulase obtained from a marine A. niger, a cellulase with endoglucanase and exoglucanase activity was efficiently expressed by constructing a vector with promoter glaA. Exoglucanase and endoglucanase activities increased from 0.21 and 4.51 U/ml of the original strain to 0.89 U/ml and 15.12 U/ml of the transformant, respectively. Filter paper activity (FPA) increased by 7.1 folds from 0.63 to 4.47 U/ml. The release of glucose by hydrolysis of wheat straw with cellulase from the transformant was 1.37 folds higher than that with cellulase from the original strain under high salinity condition. Cellulase with endoglucanase and exoglucanase activities could be well expressed in marine A. niger. The cellulase from the transformant not only showed higher activity, but also retained halostability. An appreciate proportion of β-glucosidase, exoglucanase, endgolucanasein cellulase was important for hydrolyzing cellulose.     

Analysis of the factors that affect the distribution and abundance of three Neobuxbaumia species (Cactaceae) that differ in their degree of rarity

We studied three species of columnar cacti in the genus Neobuxbaumia which differ in their degree of rarity: Neobuxbaumia macrocephala (the rarest), Neobuxbaumia tetetzo (intermediate), and Neobuxbaumia mezcalaensis (the most common). To investigate the ecological factors that limit their distribution and abundance, we surveyed 80 localities within the region of Tehuacan-Cuicatlán, in Central Mexico. At each locality we measured several environmental variables, and the density of the Neobuxbaumia populations present. We used a principal component analysis (PCA) to identify the factors that are associated to the presence/absence of each species. Additionally, we carried out multiple regressions between environmental variables and population density to test whether the variation in these variables was related to changes in abundance. The results show that factors significantly affecting the distribution of these species are mean annual temperature, altitude, rainfall, and soil properties such as texture and organic matter content. N. mezcalaensis reaches maximum population densities of 14,740 plants per ha (average density = 3943 plants per ha) and is associated with localities with relatively abundant rainfall. N. tetetzo shows maximum population densities of 14,060 plants per ha (average = 3070 plants per ha), and is associated with sites located at high latitudes and with high phosphorous content in the soil. The rarest species, N. macrocephala, shows maximum densities of 1180 plants per ha (average = 607 plants per ha) and is associated with localities with high soil calcium content. The distribution of this species is limited to sites with specific values of the environmental variables recorded, conferring it a high habitat specificity which accounts for its rarity.