Mining new resources for grape resistance against Botryosphaeriaceae: a focus on Vitis vinifera subsp. sylvestris

Botryosphaeria dieback is an important grapevine trunk disease with global impact. Susceptibility differences between grape varieties manifest as different expression of canopy symptoms in the field. However, the cause of these dieback symptoms and their relation with wood necrosis remain only partially understood. As a first step towards future strategies for resistance breeding, wood necrosis was investigated over a large selection of the Vitaceae family members following artificial inoculation of the Botryosphaeriaceae fungi Neofusicoccum parvum and Diplodia seriata into woody internodes. Large variation of resistance levels was found, with good performance in several accessions from V. vinifera subsp. sylvestris, the ancestor of cultivated grapevine. To get insight into the mechanisms of this apparent resistance, expression of defence genes was studied in V. vinifera cv. Chardonnay, Gewürztraminer and different V. vinifera subsp. sylvestris genotypes, in both green and necrotic areas of inoculated woods. Resistance to Botryosphaeriaceae in V. vinifera subsp. sylvestris correlated with earlier and higher induction of some defence genes, both in green and necrotic wood. Moreover, leaves of several V. vinifera subsp. sylvestris accessions were also less susceptible to necrosis induced by treatment with a culture filtrate of Botryosphaeriaceae, compared to commercial cultivars of V. vinifera. The results show that V. vinifera subsp. sylvestris provides interesting genetic resources for breeding new varieties with enhanced resistance to botryosphaeria dieback.     

Susceptibility of grapevine pruning wounds to infection by Lasiodiplodia theobromae and Neofusicoccum parvum

The susceptibility of 1‐ and 2‐year‐old grapevine wood to botryosphaeria canker caused by Lasiodiplodia theobromae and Neofusicoccum parvum was evaluated in California in two seasons. In the 2007 dormant season, pruning‐wound susceptibility was highest when wounds were inoculated immediately after pruning in December (80% of pruning wounds were infected in Chardonnay for both fungal species and 75% and 98% in Cabernet Sauvignon for N. parvum and L. theobromae, respectively). In the 2008 dormant season, pruning‐wound susceptibility was highest in November in Chardonnay (86% and 93% for N. parvum and L. theobromae, respectively) and in December in Cabernet Sauvignon (71% and 75% for N. parvum and L. theobromae, respectively). The lowest infection rates (13–35%) were observed when vines were pruned and inoculated in March in both dormant seasons and for both cultivars. Susceptibility of pruning wounds did not differ significantly (P = 0·7612) between 1‐ and 2‐year‐old wood and consequently, pruning‐wound protection treatments should be applied to all wounds. In conclusion, grapevine pruning wounds were susceptible to infection by L. theobromae and N. parvum to varying extents throughout the dormant season in California (November–March), but, overall, susceptibility of pruning wounds was highest when inoculations were done immediately after pruning and decreased significantly as the interval between pruning and inoculation increased. Results of this study suggest that pruning grapevines in late winter (March) in California would significantly reduce the risk of infection by L. theobromae and N. parvum.     

Co‐infection by Botryosphaeriaceae and Ilyonectria spp. fungi during propagation causes decline of young grafted grapevines

Decline of newly planted, grafted grapevines is a serious viticultural problem worldwide. In the Riverina (New South Wales, Australia), characteristic symptoms include low fruit yields, very short shoots and severely stunted roots with black, sunken, necrotic lesions. To determine the cause, roots and wood tissue from affected plants in 20 vineyards (Vitis vinifera cv. Chardonnay grafted to V. champini cv. Ramsey rootstock) were assayed for microbial pathogens. Ilyonectria spp. (I. macrodidyma or I. liriodendra, producers of phytotoxin brefeldin A, BFA, and cause of black foot disease of grapevines) and Botryosphaeriaceae spp. (predominantly Diplodia seriata) were isolated from rootstocks of 100 and 95% of the plants, respectively. Togninia minima and Phaeomoniella chlamydospora (cause of grapevine Petri disease) were isolated from 13 and 7% of affected plants, respectively. All Ramsey rootstock stems of grafted plants sampled from a supplier nursery were infected with Ilyonectria spp. and D. seriata. Diplodia seriata, but not Ilyonectria spp., was also isolated from 25% of canes sampled from the rootstock source block. Root inoculation of potted, disease‐free Chardonnay plants with Ilyonectria isolates from diseased vineyards caused typical disease symptoms, while co‐inoculation with Botryosphaeriaceae spp. increased disease severity. This is the first study to show that a major cause of young grapevine decline can be sequential infection by Botryosphaeriaceae from rootstock cuttings and Ilyonectria spp. from nursery soil. Although the Petri disease fungi were less common in young declining grafted grapevines in the Riverina, they are likely to contribute to the decline of surviving plants as they mature.     

Dynamics of post‐harvest pathogens Neofabraea spp. and Cadophora spp. in plant residues in Dutch apple and pear orchards

Post‐harvest diseases of apple and pear cause significant losses. Neofabraea spp. and Cadophora spp. infect fruits during the growing season and remain quiescent until disease symptoms occur after several months in storage. Epidemiological knowledge of these diseases is limited. TaqMan PCR assays were developed for quantification of N. alba, N. perennans, C. malorum and C. luteo‐olivacea in environmental samples. Various host tissues, dead weeds and grasses, soil and applied composts were collected in 10 apple and 10 pear orchards in May 2012. Neofabraea alba was detected in 73% of samples from apple orchards and 48% from pear orchards. Neofabraea perennans was present in a few samples. Cadophora luteo‐olivacea was detected in 99% of samples from apple orchards and 93% from pear orchards, whilst C. malorum was not detected in any sample. In apple orchards, highest concentrations of N. alba were found in apple leaf litter, cankers and mummies, and of C. luteo‐olivacea in apple leaf litter, mummies and dead weeds. In pear orchards, N. alba and C. luteo‐olivacea were found in highest concentrations in pear leaf litter and in dead weeds. Substrate colonization varied considerably between orchards. The temporal dynamics of pathogens was followed in four apple orchards and four pear orchards. In apple orchards the colonization by pathogens decreased from April until August and increased from September until December. This pattern was less pronounced in pear. Knowledge on population dynamics is essential for the development of preventative measures to reduce risks of fruit infections during the growing season.     

Host suitability of Vitis rootstocks to root‐knot nematodes (Meloidogyne spp.) and the dagger nematode Xiphinema index,and plant damage caused by infections

The host suitability of commercial Vitis rootstocks commonly used in Spain (161‐49C, 41B, 1103P, 110R, 140Ru and SO4) to root‐knot nematodes (Meloidogyne arenaria, M. incognita, M. javanica) and Xiphinema index, and damage caused by nematode infection were determined under controlled conditions. The three root‐knot nematodes reproduced with a rate higher than one in all rootstocks, indicating that they are suitable hosts for these nematodes. Growth of rootstocks infected with the root‐knot nematodes was less vigorous than that of nematode‐uninfected controls in the majority of the rootstocks studied. Root infection resulted in moderate to severe root galling in all rootstocks. The shoot and main stem diameters appeared to be the most sensitive variables of damage caused by infection by Meloidogyne spp., with reduction rates from 36% and 53% in 161‐49C to 57% and 66% in 140Ru, respectively. The shoot height was not significantly affected by the root‐knot nematodes and the root fresh weight generally increased as a consequence of intensive galling. The nematode X. index caused significant root damage with a reproduction factor higher than one in all rootstocks. However, reproduction factor was significantly influenced by the rootstock and significantly decreased by about 12‐fold (5·7 to 18·1‐fold) with the increase in inoculum density from 100 to 1000 nematodes per plant. The root dry weight was reduced by X. index infections, and was the plant growth variable most affected by the nematode infection in all rootstocks at both inoculum densities. Meloidogyne arenaria, M. incognita, M. javanica and X. index, prevalent in many world vineyards, are all shown to have a damaging effect on the six tested rootstocks.     

Post‐infection development and histopathology of Meloidogyne arenaria race 1 on Arachis spp.

The reproductive behaviour of the root‐knot nematode Meloidogyne arenaria race 1 was compared on two wild species of Arachis (A. duranensis and A. stenosperma) and cultivated peanut (A. hypogaea cv. IAC‐Tatu‐ST). The three species were considered moderately susceptible, resistant, and susceptible, respectively. Penetration and development of the root‐knot nematode in the resistant species was reduced in comparison with that occurring in susceptible plants. Several cell features, including dark blue cytoplasm and altered organelle structure were observed in the central cylinder of A. stenosperma, indicating a hypersensitive‐like response (HR) of infested host cells. Neither giant cells, nor nematodes developed beyond the second stage, were found on A. stenosperma. Arachis duranensis showed a delay in the development of nematodes in the roots compared to A. hypogaea. The two wild peanut species were chosen to be the contrasting parents of a segregating population for mapping and further investigation of resistance genes.     

Development of isolate‐specific markers for Neofusicoccum parvum and N. luteum and their use to study rainwater splash dispersal in the vineyard

Unique bands were identified in single isolates of Neofusicoccum parvum and Neofusicoccum luteum using universally primed polymerase chain reaction (UP‐PCR) analysis of isolates obtained from grapevines and non‐grapevine hosts in New Zealand, Australia, South Africa and the USA. Primers were designed to amplify a 1550 bp portion of the 1573 bp marker band from N. parvum isolate B2141 and a 510 bp portion of the 524 bp marker band from N. luteum isolate G51a2. A PCR‐RFLP assay was developed to distinguish the N. parvum isolate B2141 from other N. parvum isolates, based on a polymorphism found in the marker band using the TaqI restriction endonuclease. For N. luteum isolate G51a2, the designed primers were specific at an annealing temperature of 63°C in the PCR. The sensitivity threshold of the N. parvum and N. luteum isolate‐specific markers was 50 pg and 5 pg, respectively, when used in standard PCR with purified genomic DNA. The sensitivity of the N. parvum isolate‐specific marker was increased to 0·5 pg by nested PCR. The specificity test of both isolate‐specific markers with six other Botryosphaeriaceae spp. showed that they were specific to their respective species and isolates. Both markers were able to detect the conidia of N. parvum and N. luteum marker isolates in rainwater samples collected at different distances from an inoculation point in the vineyard. The results showed that rain splash could disperse the conidia of both of these species up to 2 m from the inoculum point in a single rainfall event.     

Sensitivity of recently naturalised Digitaria spp. populations to 4‐hydroxyphenyl pyruvate dioxygenase‐ and acetolactate synthase‐inhibiting herbicides in maize

Until recently, Digitaria aequiglumis var. aequiglumis, native to South America, and Digitaria ciliaris subsp. nubica, native to Northeast Africa, were completely overlooked in Belgium due to their close morphological resemblance to Digitaria sanguinalis and Digitaria ischaemum. One of the possible reasons for their expansion in maize fields, besides for example the lack of crop rotation, might be a lower sensitivity to post‐emergence herbicides acting against panicoid grasses. Dose – response pot experiments were conducted in the glasshouse to evaluate the effectiveness of four foliar‐applied HPPD‐inhibiting herbicides (mesotrione, sulcotrione, tembotrione, topramezone) and two foliar‐applied ALS‐inhibiting herbicides (foramsulfuron, nicosulfuron) for controlling Belgian populations of D. aequiglumis and D. ciliaris subsp. Nubica, as well as local D. sanguinalis and D. ischaemum populations. In another dose–response pot experiment, the influence of growth stage at time of herbicide application on efficacy of topramezone and nicosulfuron for Digitaria spp. control was evaluated. In general, D. aequiglumis and D. ciliaris subsp. nubica populations were less sensitive to HPPD inhibitors than D. ischaemum and D. sanguinalis populations, except for D. aequiglumis treated with topramezone. Contrary to other herbicides tested, topramezone adequately controlled all D. aequiglumis populations at doses well below maximum authorised field dose. All species tested showed a progressive decrease in sensitivity to topramezone and nicosulfuron with seedling age. A satisfactory post‐emergence control of Digitaria species in the field will require appropriate choice of herbicide and dose, as well as more timely application.     

Multiple resistance of Papaver rhoeas L. to 2,4‐D and acetolactate synthase inhibitors in four European countries

The issue of cross‐ or multiple resistance to acetolactate synthase (ALS) inhibitors and the auxinic herbicide 2,4‐D was investigated in Papaver rhoeas L., a common and troublesome weed in winter cereals, in a broad‐scale study across four European countries. A combination of herbicide sensitivity bioassays and molecular assays targeting mutations involved in resistance was conducted on 27 populations of P. rhoeas originating from Greece (9), Italy (5), France (10) and Spain (3). Plants resistant to the field rate of 2,4‐D were observed in 25 of the 27 populations assayed, in frequencies ranging from 5% to 85%. Plants resistant to ALS‐inhibiting herbicides (sulfonylureas) were present in 24 of the 27 populations, in frequencies ranging from 4% to 100%. Plants resistant to 2,4‐D co‐occurred with plants resistant to sulfonylureas in 23 populations. In four of these, the probability of presence of plants with cross‐ or multiple resistance to 2,4‐D and sulfonylureas was higher than 0.5. ALS genotyping of plants from the field populations or of their progenies, identified ALS alleles carrying a mutation at codon Pro197 or Trp574 in 2,4‐D‐sensitive and in 2,4‐D‐resistant plants. The latter case confirmed multiple resistance to 2,4‐D and ALS inhibitors at the level of individual plants in all four countries investigated. This study is the first to identify individual plants with multiple resistance in P. rhoeas, an attribute rarely assessed in other weed species, but one with significant implications in designing chemical control strategies.     

Detection of Dickeya spp. latent infection in potato seed tubers using PCR or ELISA and correlation with disease incidence in commercial field crops under hot‐climate conditions

Over a 5‐year period (2006–2010), 277 certified, visually healthy potato seed lots, imported from Europe to Israel for commercial use, were tested for Dickeya spp. latent infection by PCR analysis (277 seed lots) and ELISA (154 seed lots). Seeds from these lots were grown in commercial potato fields which were inspected twice a season by Plant Protection and Inspection Services (PPIS). Stem samples were tested for the presence of Dickeya spp. by PCR analysis. PCR and ELISA results from seed lot testing correlated with disease expression in 74 and 83·8% of the cases, respectively. Positive laboratory results with no disease symptoms in the field (‘+lab/?field’ results) comprised 24·7 and 9·7% of the PCR and ELISA analyses, respectively, whereas negative laboratory results with disease symptoms in the field results (‘?lab/+field’) were obtained in 1·3 and 6·5%, of cases respectively. Maximum disease incidence, as well as the number of cultivars expressing disease symptoms, increased over the years of this study, indicating an increase in the prevalence of the disease. Severe disease incidence was observed on cvs Dita, Rodeo, Desiree, Mondial, Tomensa and Jelly. Of the 55 imported seed lots from which disease was recorded in the field, 49 originated from the Netherlands, four from Germany and two from France. None originated from Scotland.     

A SNaPshot assay for the rapid and simple detection of known point mutations conferring resistance to ACCase‐inhibiting herbicides in Lolium spp.

Evolution of resistance to herbicides in weeds is becoming an increasing problem worldwide. To develop effective strategies for weed control, a thorough knowledge of the basis of resistance is required. Although non‐target‐site‐based resistance is widespread, target site resistance, often caused by a single nucleotide change in the gene encoding the target enzyme, is also a common factor affecting the efficacies of key herbicides. Therefore, fast and relatively simple high‐throughput screening methods to detect target site resistance mutations will represent important tools for monitoring the distribution and evolution of resistant alleles within weed populations. Here, we present a simple and quick method that can be used to simultaneously screen for up to 10 mutations from several target site resistance‐associated codons in a single reaction. As a proof of concept, this SNaPshot multiplex method was successfully applied to the genotyping of nine variable nucleotide positions in the CT domain of the chloroplastic ACCase gene from Lolium multiflorum plants from 54 populations. A total of 10 nucleotide substitutions at seven of these nine positions (namely codons 1781, 1999, 2027, 2041 2078, 2088 and 2096) are known to confer resistance to ACCase‐inhibiting herbicides. This assay has several advantages when compared with other methods currently in use in weed science. It can discriminate between different nucleotide changes at a single locus, as well as screening for SNPs from different target sites by pooling multiple PCR products within a single reaction. The method is scalable, allowing reactions to be carried out in either 96‐ or 384‐well plate formats, thus reducing work time and cost.     

Effects of resistance of Eremocitrus glauca and Microcitrus australis to viroid infection: replication,accumulation and long‐distance movement of six citrus viroids

In studies to identify genotypes resistant to infection with citrus viroids, Eremocitrus glauca and Microcitrus australis were selected because their evolution in their habitat in Australia and New Guinea may have led to the selection of unusual traits. The movement and accumulation of Citrus exocortis viroid (CEVd), Hop stunt viroid, Citrus bent leaf viroid, Citrus dwarfing viroid, Citrus bark cracking viroid and Citrus viroid V (CVd‐V) in self‐rooted as well as in graft‐ propagated E. glauca and M. australis plants was assessed by northern hybridization, RT‐PCR and by topworking to the sensitive selection 861‐S1 of Etrog citron. In both plant species the inoculated viroids were undetectable unless these plants were grafted to a susceptible Citrus partner, the rough lemon rootstock and/or the topworked Etrog citron, which acted as viroid sources. The results obtained indicate that M. australis and in particular E. glauca are poor viroid hosts in which viroid replication/accumulation does not occur or is extremely inefficient. However, viroid downward and upward movement to grafted Citrus partners in which viroid replication and accumulation occurs efficiently was not impaired. Eremocitrus glauca and M. australis showed differences regarding their properties as viroid hosts, but for both species CEVd seemed to have the lowest affinity among the viroid species tested and CVd‐V the highest. Even though E. glauca and M. australis do not appear to be truly resistant to viroid infection, they are interesting genotypes for further characterization of the mechanisms involved in viroid infection.