Putative new species of the genus Dickeya as major soft rot pathogens in Phalaenopsis orchid production

Bacterial soft rots are a serious limitation to the production of orchids and other horticultural plants. Here, the characterization of causative bacteria isolated from Phalaenopsis orchids showing symptoms, from a commercial production site, is reported. The most commonly isolated bacteria were identified as Dickeya spp. Partial sequencing of 16S rDNA, fliC and dnaX showed diversity among the isolates and divided the isolates into two groups, with greatest similarity to previously reported undefined Dickeya lineages from orchids (UDL‐3 and UDL‐4). Two isolates (B16, S1) were sequenced using next‐generation sequencing, which has provided draft genomes of these two isolates for further studies (Ali? et al., 2015 ). Newly developed fliC‐based lineage‐specific quantitative real‐time PCR assays were used to distinguish among the lineages and to assess their relative abundances in diseased tissues. Virulence and aggressiveness comparison tests in vivo on Phalaenopsis orchids, potato plants and witloof chicory leaves indicated high virulence and extreme maceration potential of these novel Dickeya isolates, compared to a reference panel of other Dickeya spp. Pantoea cypripedii (formerly Pectobacterium cypripedii), which has previously been reported as a soft rot pathogen of orchids, was not detected, and isolates obtained from culture collections did not cause symptoms on artificially infected Phalaenopsis orchids.     

Phytophthora pachypleura sp. nov., a new species causing root rot of Aucuba japonica and other ornamentals in the United Kingdom

Isolates of an unknown Phytophthora species from the ‘Phytophthora citricola complex’ have been found associated with mortality of Aucuba japonica in the UK. Based on morphological characteristics, growth–temperature relationships, sequences of five DNA regions and pathogenicity assays, the proposed novel species is described as Phytophthora pachypleura. Being homothallic with paragynous antheridia and semipapillate sporangia, P. pachypleura resembles other species in the ‘P. citricola complex’ but can be discriminated by its distinctively thick‐walled oospores with an oospore wall index of 0·71. In the phylogenetic analysis based on three nuclear (ITS, β‐tubulin, EF‐1α) and two mitochondrial (cox1, nadh1) DNA regions, P. pachypleura formed a distinct clade within the ‘P. citricola complex’ with P. citricola s. str., P. citricola E and P. acerina as its closest relatives. Phytophthora pachypleura is more aggressive to A. japonica than P. plurivora and P. multivora and has the potential to affect other ornamental species.     

Brachypodium distachyon is a pathosystem model for the study of the wheat disease rhizoctonia root rot

Brachypodium distachyon (Bd) is increasingly being used as a model for cereal diseases and to study cereal root architecture. Rhizoctonia solani AG 8 is a necrotrophic root pathogen that infects wheat soon after germination resulting in reduced plant growth and yield loss. Genetic resistance to R. solani AG 8 is not available in commercial wheat cultivars, although some quantitative levels of resistance have previously been found in mutant lines and grass relatives. Resistance mechanisms in cereals remain unknown. The ability to use Bd as a model to study the wheat–R. solani AG 8 pathosystem was investigated. The results presented show that Bd is susceptible to R. solani AG 8 and that the pathogen infects both species to a similar degree, producing comparable disease symptoms. Root length reduction was the primary indicator of disease, with shoots also affected. The second objective was to develop a repeatable phenotyping method to screen Bd populations for resistance to R. solani AG 8. Results of a preliminary experiment provide evidence for variation in resistance between Bd inbred lines. This is the first report showing the potential of Bd as a model plant for discovery of quantitative genetic variation in resistance to a necrotrophic cereal root pathogen.     

Testing and modelling the effects of climate on the incidence of the emergent nut rot agent of chestnut Gnomoniopsis castanea

Gnomoniopsis castanea is an emerging fungal pathogen causing nut rot of sweet chestnut, Castanea sativa. This study was aimed at testing and modelling the effects of climate on disease incidence. Up to 120 ripe nuts were collected in 2011 from trees in each of 12 sites located in the northwest of Italy. The incidence of G. castanea in each site was expressed as the number of infected nuts out of the total number of nuts sampled (%), determined by combining the results of morphological identification of isolates obtained from nuts, and their typing through a newly developed taxon‐specific molecular assay. Disease incidence ranged from 20 to 93%, depending on site. Geostatistical analyses revealed that, despite the clustering of sites (P < 0·05), disease incidence was not spatially autocorrelated (P > 0·05). This finding suggests that the disease is influenced by site‐dependent factors whose scale (c. 7·5–15·6 km) is consistent with the climate variability throughout the sampling region. Multivariate analyses on maximum, mean and minimum temperatures and on rainfall showed that warmer temperatures were associated with higher levels of disease incidence. The temperatures of months before nut harvesting were selected as predictors for partial least squares regression (PLSR) models (GnoMods) of G. castanea incidence. External validation on data collected either on sites or in years not used for model fitting showed the good predictive abilities of the GnoMods (Spearman's ρ_obs/pred > 0·72, P < 0·05). The above findings support a relationship between climate and incidence of G. castanea, providing statistical tools to forecast disease incidence at site level.     

Stem and root rot of scarlet runner bean (Phaseolus coccineus) caused by Pythium myriotylum

A severe rot was found on the stems and roots of scarlet runner bean (Phaseolus coccineus) in Ibaraki Prefecture (Japan) in August 2004. The causal fungus was identified as Pythium myriotylum. We propose the name of stem and root rot of scarlet runner bean (“Kuki-negusare-byo” in Japanese) for this new disease.     

The control of sclerotinia stem rot on oilseed rape (Brassica napus): current practices and future opportunities

Sclerotinia stem rot (SSR) caused by the phytopathogenic fungus Sclerotinia sclerotiorum is a major disease of oilseed rape (Brassica napus). During infection, large, white/grey lesions form on the stems of the host plant, perturbing seed development and decreasing yield. Due to its ability to produce long‐term storage structures called sclerotia, S. sclerotiorum inoculum can persist for long periods in the soil. Current SSR control relies heavily on cultural practices and fungicide treatments. Cultural control practices aim to reduce the number of sclerotia in the soil or create conditions that are unfavourable for disease development. These methods of control are under increased pressure in some regions, as rotations tighten and inoculum levels increase. Despite their ability to efficiently kill S. sclerotiorum, preventative fungicides remain an expensive gamble for SSR control, as their effectiveness is highly dependent on the ability to predict the establishment of microscopic infections in the crop. Failure to correctly time fungicide applications can result in a substantial cost to the grower. This review describes the scientific literature pertaining to current SSR control practices. Furthermore, it details recent advances in alternative SSR control methods including the generation of resistant varieties through genetic modification and traditional breeding, and biocontrol. The review concludes with a future directive for SSR control on oilseed rape.     

Resistance to Sclerotinia sclerotiorum in wild Brassica species and the importance of Sclerotinia subarctica as a Brassica pathogen

Brassica crops are of global importance, with oilseed rape (Brassica napus) accounting for 13% of edible oil production. All Brassica species are susceptible to sclerotinia stem rot caused by Sclerotinia sclerotiorum, a generalist fungal pathogen causing disease in over 400 plant species. Generally, sources of plant resistance result in partial control of the pathogen although some studies have identified wild Brassica species that are highly resistant. The related pathogen S. subarctica has also been reported on Brassica but its aggressiveness in relation to S. sclerotiorum is unknown. In this study, detached leaf and petiole assays were used to identify new sources of resistance to S. sclerotiorum within a wild Brassica ‘C genome’ diversity set. High‐level resistance was observed in B. incana and B. cretica in petiole assays, whilst wild B. oleracea and B. incana lines were the most resistant in leaf assays. A B. bourgeai line showed both partial petiole and leaf resistance. Although there was no correlation between the two assays, resistance in the detached petiole assay was correlated with stem resistance in mature plants. When tested on commercial cultivars of B. napus, B. oleracea and B. rapa, selected isolates of S. subarctica exhibited aggressiveness comparable to S. sclerotiorum indicating it can be a significant pathogen of Brassica. This is the first study to identify B. cretica as a source of resistance to S. sclerotiorum and to report resistance in other wild Brassica species to a UK isolate, hence providing resources for breeding of resistant cultivars suitable for Europe.     

Forecasting the spread of aerially transmitted crop diseases with a binary classifier for inoculum survival

The risk of between‐field spread of disease is typically omitted from crop disease warning systems, as it is difficult to know the number and location of inoculum sources and thus predict the abundance of inoculum arriving at healthy crops. This study explores the utility of a simple approach to predicting risk of between‐field spread, based on the estimated probability that inoculum will survive the transportation process. Using potato late blight as a case study, the effect of solar radiation on the viability of detached Phytophthora infestans sporangia was assessed. A model to estimate the probability of spore survival was derived using a binomial generalized linear mixed model (GLMM), and receiver operating characteristic (ROC) curve analysis and cross‐validation were used to evaluate the global performance of the model as a binary classifier for discriminating between viable and nonviable sporangia. The model yielded an area under the ROC curve of 0.92 (95% CI = 0.90–0.93), signifying an excellent classification algorithm. Inspection of the curve provided a number of suitable decision threshold (or cut‐off) probabilities for discriminating between viable and nonviable sporangia. The classifier was tested as a forecasting system for potato late blight outbreaks using 10 years of outbreak data from across Great Britain. There was a marked differentiation among the cut‐offs, but the best prediction outcome was an accuracy of 89% with an alert frequency of 1 in 7 days. This model can be easily modified or the methodology replicated for other pathosystems characterized by airborne inoculum.     

Testing and modelling the potential of three diploid plants in Poaceae as a new pathosystem to investigate the interactions between cereal hosts and cereal cyst nematode (Heterodera avenae)

Cereal cyst nematode (CCN), Heterodera avenae, is one of the most important pathogens of wheat worldwide, and causes significant yield losses. Research on CCN–wheat interactions is hampered by the lack of an effective model pathosystem. This study investigated the potential of the model cereal Brachypodium distachyon (Bd21‐3) and diploid wheat 2A (G1812) and 2D (AL8/78) as model hosts for CCN. Nematode infection analysis showed that although some CCN penetrated Bd21‐3 roots, these nematodes failed to develop to the later developmental stages or form cysts, indicating B. distachyon is not a host for CCN. A strong burst of reactive oxygen species (ROS) within Bd21‐3 roots infected with CCN was induced 3 days after infection and the expression of seven ROS‐producing genes was significantly increased. In contrast, CCN completed its life cycle in both diploid wheat 2A and 2D, and formed normal syncytia in these hosts. Although CCN developmental processes within both diploid wheat 2A and 2D were very similar to those in the susceptible control, the number of cysts formed on diploid wheat 2D was less than those formed on diploid wheat 2A and the susceptible control, indicating that diploid wheat 2A was a more suitable host for CCN than 2D. This is the first report of a potential new pathosystem for CCN–host interactions using diploid wheat.     

Use of nitrate non‐utilizing (nit) mutants to determine phenological stages at which Botrytis cinerea infects wine grapes causing botrytis bunch rot

Botrytis bunch rot (BBR), caused by Botrytis cinerea, degrades wine grapes during ripening, even though infection can occur as early as flowering. Effective BBR management requires knowledge of whether some stages of fruit development are more important than others in relation to infection and BBR severity at harvest. Bunches of Vitis vinifera ‘Sauvignon blanc’ and/or ‘Pinot noir’ were inoculated in two vineyard trials and one glasshouse trial with nitrate non‐utilizing (nit) mutant strains at three phenological stages: early flowering, pre‐bunch closure (PBC) and veraison. Isolates recovered from symptomless berries at veraison and from bunches with symptoms at harvest were screened to measure the incidence of the nit strains used in the inoculations. It was found that latent infections, which resulted in BBR at harvest, could become established at all three phenological stages and no single stage was associated with greater latent incidence or harvest severity than any other stage. It was concluded that a proportion of BBR at harvest resulted from the expression of latent infections that had accumulated throughout the season. However, the time between infection and BBR symptom expression in near‐ripe grape berries was sufficiently short for polycyclic secondary infection to also contribute to epidemic development.     

Molecular characterization,comparison of screening methods,and evaluation of cross‐pathogenicity of black rot (Xanthomonas campestris pv. campestris) strains from cabbage,choy sum,leafy mustard and pak choi from Taiwan

Choy sum (Brassica rapa var. parachinensis), leafy mustard (Brassica juncea) and pak choi (B. rapa var. chinensis) are highly nutritious components of diets in Taiwan and other Asian countries, and bacterial black rot caused by Xanthomonas campestris pv. campestris (Xcc) is a major biotic constraint in these crops. As very little was known about the Xcc strains from these crops in these regions, including their cross‐pathogenicity and aggressiveness on different hosts, Xcc strains were obtained from cabbage (Brassica oleracea var. capitata), choy sum, leafy mustard and pak choi crops in Taiwan. Two previously published PCR‐based assays reliably distinguished the Xcc strains from other Xanthomonas species and subspecies. Phylogenetic analysis based on repetitive sequence‐based PCR assays placed the Xcc strains in a clade distinct from other Xanthomonas species, and also showed host specificity. Although all of the Xcc strains from the different host species were pathogenic on all five Brassica test species in both a detached leaf assay and an intact plant assay, in the intact plant assay they showed differences in virulence or aggression on the different test hosts. The Xcc strains from leafy mustard and pak choi were consistently highly aggressive on all the test host genotypes, but the strains from choy sum and cabbage were less aggressive on leafy mustard and choy sum. The intact plant assay proved more discriminating and reliable than the detached leaf assay for comparing the aggressiveness of Xcc strains on different host genotypes, and so, with the new Xcc strains isolated in this study, will be useful for screening leafy brassica germplasm accessions for resistance to black rot.     

Molecular,morphological and pathogenic diversity of Sclerotinia sclerotiorum isolates from common bean (Phaseolus vulgaris) fields in Argentina

White mould, caused by Sclerotinia sclerotiorum, is one of the most threatening fungal diseases occurring across major bean production regions worldwide. In Argentina, under favourable weather conditions, up to 100% seed yield losses occur on susceptible common bean cultivars. The aim of this study was to characterize the diversity of S. sclerotiorum isolates from six dry bean fields in the main production area of Argentina by means of molecular, morphological (mycelium colour, number and pattern of sclerotia distribution) and pathogenic approaches. Among 116 isolates analysed, high genotypic and morphological variability was observed. A total of 52 mycelial compatibility groups (MCGs) and 59 URPs (universal rice primers) molecular haplotypes were found. All the MCGs were location specific, while only 12% of the URP haplotypes were shared among locations. The molecular analysis of variance revealed a significant differentiation among populations, with higher genetic variability within the populations analysed than among them. The aggressiveness of the isolates towards bean seedlings was assessed in the greenhouse. Most of the isolates were highly aggressive, while no variation among locations was observed. The information generated in the present study provides, for the first time, information on the variability of S. sclerotiorum associated with white mould in the main common bean production area in Argentina. In addition, the findings suggest the occurrence of both clonal and sexual reproduction in the populations analysed. This work contributes to the development of sustainable management strategies in bean production aimed to minimize yield losses due to white mould.     

Non‐pathogenic rhizosphere bacteria belonging to the genera Rhizorhapis and Sphingobium provide specific control of lettuce corky root disease caused by species of the same bacterial genera

Lettuce corky root (CR) is caused by bacteria in the genera Rhizorhapis, Sphingobium, Sphingopyxis and Rhizorhabdus of the family Sphingomonadaceae. Members of this family are common rhizosphere bacteria, some pathogenic to lettuce. Sixty‐eight non‐pathogenic isolates of bacteria obtained from lettuce roots were tested for control of CR caused by Rhizorhapis suberifaciens CA1^T and FL1, and Sphingobium mellinum WI4^T. In two initial screenings, 10 isolates significantly reduced CR induced by one or more pathogenic strains on lettuce seedlings in vermiculite, while seven non‐pathogenic isolates provided significant CR control in natural or sterilized field soil. Rhizorhapis suberifaciens FL11 was effective at controlling all pathogenic strains, but most effective against R. suberifaciens CA1^T. The other selected isolates controlled only pathogenic strains belonging to their own genus. In a greenhouse experiment, a soil drench with selected biocontrol agents (R. suberifaciens FL11, Sphingomonas sp. NY3 and S. mellinum CA16) controlled CR better than seed treatments or application of alginate pellets. In microplots infested with R. suberifaciens CA1^T, seed treatment with R. suberifaciens FL11 provided complete control and a soil drench with FL11 significantly reduced the disease. Pathogenicity tests with FL11 on 23 plant species in 10 families resulted in slight yellowing on roots of lettuce and close relatives; similar yellowing appeared on some roots of non‐inoculated lettuce plants. This research showed that biocontrol agents can be genus‐specific. Only one isolate, FL11, provided more general control of various pathogenic strains causing CR even in field soil in pots and microplots.     

Investigations into Encephalartos insect pests and diseases in South Africa and identification of Phytophthora cinnamomi as a pathogen of the Modjadji cycad

South Africa holds the greatest diversity of Encephalartos species globally. In recent years several reports have been received of Encephalartos species in the country dying of unknown causes. The aim of this study was to investigate the presence of, and identify the causal agents of, diseases of Encephalartos species in the Gauteng and Limpopo Provinces of South Africa. Plant material with symptoms and insects were collected from diseased plants in private gardens, commercial nurseries and conservation areas in these regions. Insects collected were identified based on morphology, and microbial isolates based on morphology and DNA sequence data. Insect species identified infesting cultivated cycads included the beetle Amorphocerus talpa, and the scale insects Aonidiella aurantii, Aspidiotus capensis, Chrysomphalus aonidum, Lindingaspis rossi, Pseudaulacaspis cockerelli, Pseudaulacaspis pentagona and Pseudococcus longispinus. Fungal species isolated from diseased plants included species of Diaporthe, Epicoccum, Fusarium, Lasiodiplodia, Neofusicoccum, Peyronellaea, Phoma, Pseudocercospora and Toxicocladosporium. The plant pathogen Phytophthora cinnamomi was identified from E. transvenosus plants in the Modjadji Nature Reserve. Artificial inoculation studies fulfilled Koch's postulates, strongly suggesting that P. cinnamomi is responsible for the deaths of these plants under field conditions.     

Analysis of resistance to witches’ broom disease (Moniliophthora perniciosa) in flower cushions of Theobroma cacao in a segregating population

This study evaluates resistance to witches’ broom disease in flower cushions of Theobroma cacao under field conditions. The aim was to determine optimal inoculation methods to evaluate the disease incidence using flower cushions in the field. A segregating mapping population of 580 trees (cultivar TSH 1188 × CCN 51) was analysed under two field conditions: high and low inoculum levels (in different years), corresponding respectively to trees with or without dried witches’ brooms hanging on the trees and producing basidiocarps. The number of newly formed cushion brooms in each tree was counted by the conventional method, and also the healthy and infected flower cushions in three 30 cm‐long regions along the trunk and the two main branches. The field inoculation methods discriminated between genotypes, with a 26% increase in disease incidence by Moniliophthora perniciosa at high inoculum. Two different segregation patterns were also observed: 27:27:9:1 under low, and 27:9:9:9:3:3:3:1 under high inoculum potential. It was also determined that at least 20 flower cushions were needed to accurately determine the percentage of infection. These methodologies allowed identification of the extreme phenotypes in this mapping population, and can therefore facilitate the detection of sources of resistance to witches’ broom disease.     

Development of a tomato plant resistant to Clavibacter michiganensis using the endolysin gene of bacteriophage CMP1 as a transgene

The bacteriophage CMP1 endolysin gene (lys), encoding, a peptidase that was shown to effectively reduce Clavibacter michiganensis by specifically hydrolysing its murein, was transferred into tomato plants by Agrobacterium‐mediated transformation. The presence of the gene was verified by PCR and the gene product was confirmed in immunoblots and stably expressed over three generations. Transgenic tomato plants did not show disease symptoms after infection with C. michiganensis subsp. michiganensis, despite the fact that small amounts of bacteria could still be identified in xylem sap and leaf extracts, although in significantly reduced amounts.     

Distinguishing sporulating and nonsporulating lesions as a method for evaluating the resistance of sugarcane genotypes to orange rust

Sugarcane breeding programmes rank the resistance of genotypes to Puccinia kuehnii, causal agent of orange rust, according to levels of disease severity. However, during the screening stages, this method of assessment can lead to precipitous elimination of genotypes with promising agronomic traits but showing mild symptoms of rust such as flecks or lesions that do not produce spores. This study aimed to propose a new method to classify the resistance of sugarcane genotypes to orange rust by counting sporulating lesions. Five sugarcane varieties with different levels of resistance to P. kuehnii were inoculated with two pathogen populations under controlled conditions. The disease severity (SEV), total number of lesions (TNL), and total number of sporulating lesions (TNSL) were evaluated in a 20 cm leaf fragment from the most diseased leaf. The TNL and TNSL evaluations were performed at 11, 16 and 21 days after inoculation (DAI) and SEV at 21 DAI. The thresholds of 80% and 8% of sporulating lesions (SL) separated susceptible from the intermediate varieties and intermediate from the resistant ones, respectively. It is proposed that the method of counting sporulating lesions be used in screening genotypes for resistance to P. kuehnii in sugarcane breeding programmes.     

Real‐time quantitative PCR assays for the rapid detection and quantification of Fusarium oxysporum f. sp. phaseoli in Phaseolus vulgaris (common bean) seeds

Fusarium oxysporum f. sp. phaseoli (Fop) is a devastating pathogen that can cause significant economic losses and can be introduced into fields through infested Phaseolus vulgaris (common bean) seeds. Efficient seed health testing methods can aid in preventing long‐distance dissemination of this pathogen by contaminated seeds. In order to improve detection of Fop in seed, a rapid, accurate and sensitive real‐time PCR assay (qPCR) protocol was developed for detection of Fop in common bean seeds. Seed lots of seven cultivars with infection incidence ranging from 0·25 to 20% were prepared by mixing known amounts of Fop‐infected seeds with Fop‐free seeds. Direct comparisons between SYBR Green and TaqMan qPCR methods were performed using primers based on the Fop virulence factor ftf1. The primers developed in this study produced a 63 bp product for highly virulent strains of Fop but did not produce an amplicon for nonpathogenic or weakly pathogenic isolates of F. oxysporum from P. vulgaris or other hosts. Under optimized conditions, both qPCR assays detected Fop infection at low levels (0·25%); however, the results suggest the TaqMan assay was more reliable at quantification than the SYBR Green assay. Linear regression models were fitted to the relationships between results of qPCR assays and infection incidence, but the models differed among cultivars. Fungal biomass per seed differed among cultivars and was related to seed size. The results indicate that the TaqMan assay developed in this study is a useful tool for the detection and quantification of Fop in bean seeds.     

Evidence for Lettuce big‐vein associated virus as the causal agent of a syndrome of necrotic rings and spots in lettuce

Lettuce big‐vein associated virus (LBVaV, genus Varicosavirus) was shown to be responsible for characteristic necrotic symptoms observed in combination with big‐vein symptoms in lettuce breeding lines when tested for their susceptibility to lettuce big‐vein disease (BVD) using viruliferous Olpidium virulentus spores in a nutrient film technique (NFT) system. Lettuce plants showing BVD are generally infected by two viruses: Mirafiori lettuce big‐vein virus (MiLBVV, genus Ophiovirus) and LBVaV. New mechanical inoculation methods were developed to separate the two viruses from each other and to transfer both viruses to indicator plants and lettuce. After mechanical inoculation onto lettuce plants MiLBVV induced vein‐band chlorosis, which is the characteristic symptom of BVD. LBVaV caused a syndrome of necrotic spots and rings which was also observed earlier in lettuce plants inoculated in the NFT system, resembling symptoms described for lettuce ring necrosis disease (RND). This observation is in contrast with the idea that LBVaV only causes latent infections in lettuce. De novo next‐generation sequencing demonstrated that LBVaV was the only pathogen present in a mechanically inoculated lettuce plant with symptoms, providing evidence that LBVaV was the causal agent of the observed necrotic syndrome and thus fulfilling Koch’s postulates for this virus. The necrotic syndrome caused by LBVaV in lettuce is referred to as LBVaV‐associated necrosis (LAN).