Target site insensitivity mutations in the AChE and LdVssc1 confer resistance to pyrethroids and carbamates in Leptinotarsa decemlineata in northern Xinjiang Uygur autonomous region

A survey of resistance to five conventional insecticides was conducted in 2009 and 2010 for the first generation 4th-instar larvae of Leptinotarsa decemlineata from Urumqi, Changji, Qitai and Qapqal. Compared with the Tekes population, a reference susceptible population, the Changji and Qapqal populations exhibited very high to moderate levels of resistance to cyhalothrin and deltamethrin, moderate to high levels of resistance to carbosulfan and carbofuran, and low levels of resistance to azinphosmethyl. Moreover, the Urumqi and the Qitai populations reached a high and a moderate level of resistance to carbosulfan, respectively. Synergistic effects of triphenyl phosphate, diethylmeleate, and piperonyl butoxide on cyhalothrin and carbosulfan in Changji population revealed that cytochrome P450s were involved in the resistance to cyhalothrin but not carbosulfan. A modified bi-PASA was developed to simultaneously detect point mutations of S291G in the AChE and L1014F in the LdVssc1 genes. The former mutation resulted in the resistance to carbamates and the latter in the resistance to pyrethroids. The rates of homozygous and heterozygous resistant individuals to carbamates (S291G mutation) were 17.6% and 14.7%, 50.6% and 42.2%, 49.9% and 41.7%, 51.3% and 41.4%, and 44.8% and 47.4%; to pyrethroids (L1014F mutation) were 5.8% and 8.7%, 36.1% and 27.0%, 41.8% and 24.8%, 12.2% and 9.7%, and 7.9% and 10.6%, respectively, in samples from Tekes, Changji, Qapqal, Urumqi and Qitai. I392T point mutation in the AChE was detected by RT-PCR among 18 individuals from Changji, Qapqal, Urumqi and Qitai. These results demonstrated that point mutations of S291G in the AChE and L1014F in the LdVscc1 are responsible for, at least partially, the resistance to carbamates and pyrethroids in L. decemlineata in some field populations in northern Xinjiang Uygur autonomous region.     

Target-site resistance to pyrethroids in European populations of pollen beetle, Meligethes aeneus F.

Pollen beetle, Meligethes aeneus F. (Coleoptera: Nitidulidae) is a major univoltine pest of oilseed rape in many European countries. Winter oilseed rape is cultivated on several million hectares in Europe and the continuous use of pyrethroid insecticides to control pollen beetle populations has resulted in high selection pressure and subsequent development of resistance. Resistance to pyrethroid insecticides in this pest is now widespread and the levels of resistance are often sufficient to result in field control failures at recommended application rates. Recently, metabolic resistance mediated by cytochrome P450 monooxygenases was implicated in the resistance of several pollen beetle populations from different European regions. Here, we have also investigated the possible occurrence of a target-site mechanism caused by modification of the pollen beetle para-type voltage-gated sodium channel gene. We detected a single nucleotide change that results in an amino acid substitution (L1014F) within the domain IIS6 region of the channel protein. The L1014F mutation, often termed kdr, has been found in several other insect pests and is known to confer moderate levels of resistance to pyrethroids. We developed a pyrosequencing-based diagnostic assay that can detect the L1014F mutation in individual beetles and tested more than 350 populations collected between 2006 and 2010 in 13 European countries. In the majority of populations tested the mutation was absent, and only samples from two countries, Denmark and Sweden, contained pollen beetles heterozygous or homozygous for the L1014F mutation. The mutation was first detected in a sample from Denmark collected in 2007 after reports of field failure using tau-fluvalinate, and has since been detected in 7 out of 11 samples from Denmark and 25 of 33 samples from Sweden. No super-kdr mutations (e.g. M918T) known to cause resistance to pyrethroids were detected. The implications of these results for resistance management strategies of pollen beetle populations in oilseed rape crops are discussed.     

Seiridium cardinale and other cypress cankers1

Several species of microscopic fungi have been associated with canker diseases of wild and cultivated species of Cupressus and related genera. Seiridium cardinale was first reported in Europe (France) in 1944 and then spread to other countries, causing what at present is the major disease of cypress in Europe. Over the last two decades, heavy losses have been caused to the nursery industry, to cypress plantations used for forestation and windbreaks as well as to ornamental cypresses. The death of millions of trees has devastated the traditional and beloved landscape of areas where cypresses constitute a major element of the vegetation. Lepteutypa cupressi (anamorph Seiridium cupressi) has recently been reported in Greece (Kos). It had already caused considerable losses on cypress plantations in other geographical areas (Africa, Australia, Oceania). Some taxonomic aspects of the pathogen are discussed. The introduction of L. cupressi into the Mediterranean area, where S. cardinale has already spread, not only represents a serious threat to European cypress plantations, but also complicates the efforts made up to now towards the selection of resistant clones and the improvement of genetic resistance of the Mediterranean cypress C. sempervirens to S. cardinale. Thus, immediate action is required in order to eradicate the existing foci and to avoid further spread of the disease. Seiridium unicorne was first described in the USA in 1878 and then recorded again in North America, Europe, Asia and Oceania. The host range of this pathogen is not restricted to Cupressaceae and includes plants belonging to eight families. It seems that the canker disease due to S. unicorne is not so serious as that caused by S. cardinale or L. cupressi. L. cupressi has probably been misidentified as S. unicorne in records of epidemics in East Africa and other areas of the world.     

Real-time PCR as a promising tool to monitor growth of Venturia spp. in scab-susceptible and -resistant apple leaves

Apple scab, the most important disease of apple worldwide, is caused by Venturia inaequalis. Currently, evaluation of fungal pathogenicity and host resistance is based on a symptomatic disease rating. However, this method does not provide an accurate measurement of the degree of infection and cannot detect early fungal development in symptomless leaves. In this study, a Venturia-specific real-time PCR assay was developed using primers designed around the specific internal transcribed spacer 2 (ITS2) region of the 5.8S rRNA gene. Using SYBR? Green I technology, the assay can accurately quantify Venturia DNA over a concentration range of at least five orders of magnitude. Detection sensitivities were in the order of 100?fg. The method was used to quantify Venturia genomic DNA levels in leaves of three apple cultivars with different levels and types of scab resistance and artificially infected with V. inaequalis. The assay clearly discriminated between Venturia levels in monogenic resistant (‘Topaz’), polygenic resistant (‘Discovery’), and susceptible (‘Golden Delicious’) cultivars, and proved especially useful to quantify pathogen levels during the initial latent stage of infection. The real-time PCR data of ‘Golden Delicious’ were consistent with the observed evolution of the degree of sporulation during a time-course experiment. Although measurements were influenced by the presence of co-extracted PCR-inhibitors, the impact of these compounds was independent of the apple cultivar or the initial amount of fungal DNA present. In conclusion, real-time PCR amplification of the ITS2-5.8S rDNA of Venturia spp. is a faster, more objective and more sensitive method to monitor fungal growth and to evaluate host resistance than phenotypic disease rating scores.     

Recent outbreaks of rust diseases and the importance of basic biological research for controlling rusts

Rust fungi are obligate plant parasites belonging to the order Pucciniales; they comprise about 7,800 species throughout the world. Some species seriously damage crops, vegetables, fruits and trees. Of these species, wheat stem rust (Puccinia graminis f. sp. tritici), Asian soybean rust (Phakopsora pachyrhizi) and myrtle rust (Puccinia psidii) have recently become major concerns worldwide, and this review, discusses recent rust disease outbreaks of Asian soybean rust and myrtle rust. Both rusts have very wide host ranges. Asian soybean rust has spread from its original region of distribution (eastern Asia) to many areas of soybean cultivation around the world. Myrtle rust is a new disease in areas where host plants were first introduced and has spread to other parts of the world including the areas where the host plants are indigenous. New diseases of economically important plants can occur by host shifts from wild host plants or host jumps from phylogenetically unrelated plant species. Recent advances in molecular phylogenetic studies have contributed to a revision of rust taxonomy. Molecular phylogenetic analyses, together with precise morphological observations and inoculation experiments, have identified taxonomic groups among populations that are morphologically very similar. Systematic, ecological and other basic biological studies of rust fungi in both cultivated and wild host plants are very important for developing methods to control rust diseases. Recent changes in the International Code of Botanical Nomenclature will surely affect the systematics of rust fungi.     

Cytochrome P-based resistance mechanism and pyrethroid resistance in the field Anopheles albimanus resistance management trial

The relative rates of cytochrome P⁴⁵⁰ selection in southern Mexican Anopheles albimanus populations were investigated during a 3 years indoor residual house spraying intervention with a pyrethroid (PYR) or DDT, a mosaic of organophosphate (OP)-PYR, and the annual rotation of OP-PYR-carbamate (CARB). This insecticide resistance mechanism, initially evenly spread in the mosquito population, correlated with PYR resistance during the second treated year, when cytochrome P⁴⁵⁰ contents increased in most villages of the PYR, rotation and mosaic schemes. However, by the third year, mean cytochrome P⁴⁵⁰ contents declined to susceptible levels in mosquitoes of the rotation and one mosaic group but not in the PYR-treated villages. In DDT-treated villages, a continuous decrement of cytochrome P⁴⁵⁰ levels occurred since the first treatment year, and susceptible levels were observed at the end of the intervention. Most correlations of cytochrome P⁴⁵⁰ levels and PYR resistance were lost during the third year, indicating that another mechanism evolved in resistant mosquito populations.     

Influence of spatial structure on pathogen colonization and extinction: a test using an experimental metapopulation

The Linum marginale–Melampsora lini plant–pathogen interaction has been studied extensively with regard to its epidemiology and population genetic structure (host resistance and pathogen virulence) in a natural metapopulation. In this study, this system was used in an experimental metapopulation approach to investigate explicitly how the distance (degree of isolation) between local population patches influences disease dynamics within a growing season, as well as the genetic structure of pathogen populations through stochastic colonization and extinction processes. The experimental design centred on four replicate sets of populations, within which patches were spaced at increasingly greater distances apart. Each patch consisted of an identical set of host and pathogen genotypes, with each pathogen genotype having the ability to attack only one of four host-resistance types. Over the 2 years of the experiment, the results showed clear 'boom-and-bust' epidemic patterns, with the strongest determinant of disease dynamics within a growing season being the identity of particular host–pathogen genotypic combinations. However, there were also significant effects of spatial structure, in that more isolated patches tended to exhibit lower levels of disease during epidemic peaks than patches that were close together. Extinction of pathogen genotypes from individual populations was positively related to the severity of disease during preceding epidemic peaks, but negatively related to the level of disease present at the final census prior to overwintering. The probability of recolonization of pathotypes into populations during the second growing season was most strongly related to the distance to the nearest neighbouring source population in which a given pathotype was present. Overall, these results highlight the importance of spatial scale in influencing the numerical and genetical dynamics of pathogen populations.     

World-Wide Importance of Phoma Stem Canker (Leptosphaeria maculans and L. biglobosa) on Oilseed Rape (Brassica napus)

Phoma stem canker is an internationally important disease of oilseed rape (Brassica napus, canola, rapeseed), causing serious losses in Europe, Australia and North America. UK losses of €56M per season are estimated using national disease survey data and a yield loss formula. Phoma stem canker pathogen populations comprise two main species, Leptosphaeria maculans, associated with damaging stem base cankers, and Leptosphaeria biglobosa, often associated with less damaging upper stem lesions. Both major gene and quantitative trait loci mediated resistance to L. maculans have been identified in B. napus, but little is known about resistance to L. biglobosa. Leptosphaeria maculans, which has spread into areas in North America and eastern Europe where only L. biglobosa was previously identified, now poses a threat to large areas of oilseed rape production in Asia. Epidemics are initiated by air-borne ascospores; major gene resistance to initial infection by L. maculans operates in the leaf lamina of B. napus. It is not clear whether the quantitative trait loci involved in the resistance to the pathogen that can be assessed only at the end of the season operate in the leaf petioles or stems. In countries where serious phoma stem canker epidemics occur, a minimum standard for resistance to L. maculans is included in national systems for registration of cultivars. This review provides a background to a series of papers on improving strategies for managing B. napus resistance to L. maculans, which is a model system for studying genetic interactions between hemi-biotrophic pathogens and their hosts.     

TaqMan real-time PCR method for detection of <Emphasis Type="Italic">Discula destructiva</Emphasis> that causes dogwood anthracnose in Europe and North America

Dogwood anthracnose, caused by the asexual filamentous fungus Discula destructiva Redlin, is a disease of several Cornus L. species. First reported in the 1970s in the United States, the pathogen has devastated North American dogwood populations causing widespread loss that has greatly impacted forest ecosystems. In the early 2000s, the disease was discovered in Italy and Germany, spread probably through the ornamental nursery trade. The origin of D. destructiva in North America remains a mystery. To facilitate studies on its origin and dispersal, a fast and accurate method using real-time PCR was developed in this study to detect and quantify D. destructiva. The assay was validated with samples from the U.S., Italy, and Switzerland as well as phylogenetically closely related fungal species, and other fungi and oomycetes commonly found on Cornus. This method allows for fast and sensitive detection of D. destructiva in host tissue and should be useful in disease management and pest interception to prevent further spread of the pathogen.     

A potential perennial host for <Emphasis Type="Italic">Pseudoperonospora cubensis</Emphasis> in temperate regions

Pseudoperonospora cubensis causes great losses in cucurbitaceous crops worldwide. In cool temperate climates of northern Europe or North America overwintering as active mycelium is not possible, because all hosts so far reported there are summer annuals. Oospores have not yet been found in these regions under field conditions. The only perennial member of the Cucurbitaceae found naturally in central and northern Europe is Bryonia dioica. To date this plant has not been recorded as a host for downy mildews, but our infection trials demonstrate that P. cubensis is able to infest this plant. Amplification and sequencing of the ITS rDNA confirmed the observed downy mildew disease on B. dioica as P. cubensis. From these findings, the possibility that P. cubensis may be able to overwinter on this perennial host cannot be excluded. Whether or not B. dioica plays a part in the epidemology of P. cubensis in Europe requires evaluation by further studies.     

Pathotypic diversity of Hyaloperonospora brassicae collected from Brassica oleracea

Downy mildew caused by Hyaloperonospora brassicae is an economically destructive disease of brassica crops in many growing regions throughout the world. Specialised pathogenicity of downy mildews from different Brassica species and closely related ornamental or wild relatives has been described from host range studies. Pathotypic variation amongst Hyaloperonospora brassicae isolates from Brassica oleracea has also been described; however, a standard set of B. oleracea lines that could enable reproducible classification of H. brassicae pathotypes was poorly developed. For this purpose, we examined the use of eight genetically refined host lines derived from our previous collaborative work on downy mildew resistance as a differential set to characterise pathotypes in the European population of H. brassicae. Interaction phenotypes for each combination of isolate and host line were assessed following drop inoculation of cotyledons and a spectrum of seven phenotypes was observed based on the level of sporulation on cotyledons and visible host responses. Two host lines were resistant or moderately resistant to the entire collection of isolates, and another was universally susceptible. Five lines showed differential responses to the H. brassicae isolates. A minimum of six pathotypes and five major effect resistance genes are proposed to explain all of the observed interaction phenotypes. The B. oleracea lines from this study can be useful for monitoring pathotype frequencies in H. brassicae populations in the same or other vegetable growing regions, and to assess the potential durability of disease control from different combinations of the predicted downy mildew resistance genes.     

GC-MS metabolite profiling of <Emphasis Type="Italic">Phytophthora infestans</Emphasis> resistant to metalaxyl

Phytophthora infestans is the most important potato pathogen worldwide. Various alternatives have been used to control the pathogen, including continuous applications of phenylamide fungicides which has caused a rapid development of resistance in populations of P. infestans. Despite the importance of the disease, metabolite profiling of fungicide-resistant P. infestans has not been reported. In vitro resistance of Phytophthora infestans isolates to metalaxyl was characterized and metabolic changes in resistant isolates were evaluated at low (0.5 mg/L) and high (100 mg/L) concentrations of the fungicide. About 70% of the isolates tested showed resistance to metalaxyl and a total of 49 metabolites were differently expressed in resistant isolates growing in the presence of the fungicide. Principal components analysis revealed a distinct metabolite profile of resistant isolates exposed to both low and high levels of metalaxyl. The main metabolites responsible for the clustering in both fungicide concentrations included fatty acids such as hexadecanoic and octadecanoic acids, sugars such as glucose and fructose, aminoacids such as proline and valine, and organic acids such as butanedioic and propanoic acids. Potential resistance-related metabolic pathways are mostly involved in the regulation of the pathogen’s membrane fluidity and included the fatty acid biosynthesis as well as the glycerophospholipid metabolism pathways. This is the first metabolomic-based characterization of fungicide resistance in plant pathogens.     

Multivariate Analysis of the Agricultural Management Presence of Sorghum Halepense (L.) Pers. Relationships in Maize Crops

Sorghum halepense (L.) Pers. is one of the most troublesome weeds in warm climates. Its control is difficult, and understanding the factors affecting its spreading is crucial. A study was conducted in 47 commercial maize fields, which account for more than 400 ha in the Spanish provinces of Albacete, Badajoz and Madrid, to analyse the distribution of S. halepense as a function of various agricultural variables. The results showed significant effects of agricultural management on the presence of this weed. Crop rotation decreased the infestation of S. halepense. Furrow irrigation system favored the establishment of large patches with high plant density, while the sprinkler irrigation system favored the presence of isolated plants or small patches. Apparently, moldboard tillage promoted the establishment of large patches. The combination of different variables also had effects on the characteristics of the present infestations, and its management could lead to a better control in maize fields.     

Host-based life cycle traits and detoxification enzymes of major looper pests (Lepidoptera: Geometridae) of tea from Darjeeling Terai,India

Three polyphagous looper caterpillars, Buzura suppressaria Guenée, Hyposidra talaca Walker and Hyposidra infixaria Walker, have established themselves as severe pests of tea [Camellia sinensis (L.) Kuntze] in the plantations of sub Himalayan West Bengal (Terai region). Schima wallichii (DC.) Korth. is a naturally occurring alternative host of all these looper species. To gain an insight into looper and host plant relationships, the present work contemplates studies on host preference, host-based life cycle traits and levels of detoxification enzymes, such as general esterases (GEs) and glutathione S-transferases (GSTs). From the study, host-induction of feeding preference was evident in all the three looper species. Hyposidra spp. exhibited similar post-embryonic development periods both on tea and S. wallichii, whereas B. suppressaria reared on tea needed a longer development period than on S. wallichii. Tea-reared caterpillars of Hyposidra spp. were significantly heavier, having higher quantities of GEs and GSTs than S. wallichii-reared ones. B. suppressaria, however, exhibited similar body weights on tea and S. wallichii. While GST level was higher in tea-reared B. suppressaria, its GE quantity was higher on S. wallichii. Although tea was found to be a more suitable host for Hyposidra spp., the host S. wallichii proved marginally better than tea for supporting B. suppressaria. However, all the three looper species could utilize the foliage of tea and S. wallichii successfully. So S. wallichii trees can act as a sylvan reservoir of the looper species, prompting their possible invasion of tea plantations and thus making management of looper pests in tea more difficult.     

Acetylcholinesterase point mutations putatively associated with monocrotophos resistance in the two-spotted spider mite

Molecular mechanisms of monocrotophos resistance in the two-spotted spider mite (TSSM), Tetranychus urticae Koch, were investigated. A monocrotophos-resistant strain (AD) showed ca. 3568- and 47.6-fold resistance compared to a susceptible strain (UD) and a moderately resistant strain (PyriF), respectively. No significant differences in detoxification enzyme activities, except for the cytochrome P450 monooxygenase activity, were found among the three strains. The sensitivity of acetylcholinesterase (AChE) to monocrotophos, however, was 90.6- and 41.9-fold less in AD strain compared to the UD and PyriF strains, respectively, indicating that AChE insensitivity mechanism plays a major role in monocrotophos resistance. When AChE gene (Tuace) sequences were compared, three point mutations (G228S, A391T and F439W) were identified in Tuace from the AD strain that likely contribute to the AChE insensitivity as predicted by structure analysis. Frequencies of the three mutations in field populations were predicted by quantitative sequencing (QS). Correlation analysis between the mutation frequency and actual resistance levels (LC₅₀) of nine field populations suggested that the G228S mutation plays a more crucial role in resistance (r² = 0.712) compared to the F439W mutation (r² = 0.419). When correlated together, however, the correlation coefficient was substantially enhanced (r² = 0.865), indicating that both the F439W and G228S mutations may work synergistically. The A391T mutation was homogeneously present in all field populations examined, suggesting that it may confer a basal level of resistance.     

Diversity and frequencies of genetic mutations involved in insecticide resistance in field populations of the house fly (Musca domestica L.) from China

Insecticides have been extensively used for house fly control in China, with dichlorvos and deltamethrin being widely used. Knowledge about the current status of insecticide resistance and the underlying genetic changes is crucial for developing effective fly control strategies. The susceptibility to dichlorvos and deltamethrin, and the frequencies of genetic mutations involved in insecticide resistance were studied in five field populations of the house fly collected across China. Bioassay results show that flies exhibit 14- to 28-fold resistance to dichlorvos and 41- to 94-fold resistance to deltamethrin, indicating that dichlorvos and deltamethrin resistance are common in house fly populations in China. Molecular analysis reveals that flies from the five various locations carry resistance alleles at multiple loci and have diverse allelic types, different relative frequencies and combinations of each allele. Four non-synonymous single nucleotide polymorphisms (SNPs) (i.e. V260L, G342A/V, F407Y) in acetylcholinesterase (Ace) and two mutations (W251L/S) in a carboxylesterase (MdαE7) were commonly present in the field house flies. The L1014H rather than L1014F mutation in the voltage sensitive sodium channel gene (Vssc) was widely distributed in Chinese house flies. CYP6D1v1, which confers pyrethroid resistance, was found in all the five tested populations in China, although its frequency in house fly from Shandong province was very low. Our results suggest that resistance monitoring and management of house flies should be customized for a given location.     

Diagnostics,genetic diversity and pathogenic variation of ascochyta blight of cool season food and feed legumes

Molecular diagnostic techniques have been developed to differentiate the Ascochyta pathogens that infect cool season food and feed legumes, as well as to improve the sensitivity of detecting latent infection in plant tissues. A seed sampling technique was developed to detect a 1% level of infection by Ascochyta rabiei in commercial chickpea seed. The Ascochyta pathogens were shown to be genetically diverse in countries where the pathogen and host have coexisted for a long time. However, where the pathogen was recently introduced, such as A. rabiei to Australia, the level of diversity remained relatively low, even as the pathogen spread to all chickpea-growing areas. Pathogenic variability of A. rabiei and Ascochyta pinodes pathogens in chickpea and field pea respectively, appears to be quantitative, where measures of disease severity were based on aggressiveness (quantitative level of infection) rather than on true qualitative virulence. In contrast, qualitative differences in pathogenicity in lentil and faba bean genotypes indicated the existence of pathotypes of Ascochyta lentis and Ascochyta fabae. Therefore, reports of pathotype discrimination based on quantitative differences in pathogenicity in a set of specific genotypes is questionable for several of the ascochyta-legume pathosystems such as A. rabiei and A. pinodes. This is not surprising since host resistance to these pathogens has been reported to be mainly quantitative, making it difficult for the pathogen to overcome specific resistance genes and form pathotypes. For robust pathogenicity assessment, there needs to be consistency in selection of differential host genotypes, screening conditions and disease evaluation techniques for each of the Ascochyta sp. in legume-growing countries throughout the world. Nevertheless, knowledge of pathotype diversity and aggressiveness within populations is important in the selection of resistant genotypes.