Races of Xanthomonas campestris pv. vesicatoria overcoming the gene Bs2 for bacterial spot resistance in pepper, prevalent on Capsicum chinense in Barbados and Grenada and weakly pathogenic on bell pepper and tomato in the field

A total of 404 isolates of Xanthomonas campestris pv. vesicatoria , obtained from Capsicum chinense cv. West Indian Red grown in Barbados and Grenada, were differentiated into pathogenic races, and of these, 96 were tested also for selected taxonomic group phenotypes. The response of C. chinense to infection by several X. campestris pv. vesicatoria races and the contribution of races isolated from this cultivar to severity of bacterial spot of bell pepper and tomato were also investigated. P4T2, P5T2 and P6T2 were the predominant races of X. campestris pv. vesicatoria isolated from C. chinense grown in Grenada, whereas nine races (T1, P4, P6, P0T2, P1T2, P4T1, P4T2, P6T1 and P6T2) were isolated in Barbados. Race P4T2 comprised 46.0 and 71.4% of the isolates from Barbados and Grenada, respectively. The 96 isolates, all of which overcame resistance conferred by the gene Bs2 , shared taxonomic group B strain characteristics, including the presence of the β-protein band, positive amylolytic activity and inability to oxidize cis -aconitate. The C. chinense cv. West Indian Red was susceptible only to races of X. campestris pv. vesicatoria that can overcome Bs2 gene resistance. Of six such races identified in Barbados, only P4T1, P4T2 and P6T1 affected bacterial spot-susceptible bell pepper or tomato in the field, and they amounted to only 1.5–2.1% of each sample of isolates from these plant species. Moreover, they were confined to the smallest bacterial spot lesions. Bell pepper was most severely affected by combinations of races T1 with P3T2 and T2 with P0T1, and tomato by race T1 only and combinations of races P0T1 with P0T2 and P1T1 with P1T0, all of which prevailed in the field despite selection against them by C. chinense cv. West Indian Red.     

Epidemiology of dark leaf spot caused by Alternaria brassicicola and A. brassicae in organic seed production of cauliflower

In organic seed production of Brassica vegetables, infections by Alternaria brassicicola and A. brassicae can cause severe losses of yield and seed quality. Four field experiments with or without artificial inoculation with A. brassicicola were conducted in organically managed seed‐production crops of cauliflower cv. Opaal RZ in 2005 and 2006 in the Netherlands. The development of A. brassicicola and A. brassicae on pod tissues and developing seeds was followed and seed quality was assessed. Alternaria brassicicola was externally present on 1·2% of the seeds 14 days after flowering and observed internally within 4 weeks after flowering. In both seasons, seed colonization by the pathogen increased slowly until maturation but sharply increased during maturation. A similar pattern was found for the colonization of pod tissues by A. brassicicola as quantified by TaqMan‐PCR. The incidence of A. brassicicola on mature seeds reached 70–90%. Internal colonization was found for 62–80% of the seeds. External and internal seed colonization by A. brassicae was much lower, with incidences below 3%. The quality of harvested seeds was generally low, with less than 80% of seeds able to germinate. Seed quality was not affected by warm water treatments. It was concluded that A. brassicicola and A. brassicae have the potential to infect pods and seeds soon after flowering. For the production of high quality seeds, producers must prevent such early infections. Therefore, new control measures are needed for use in organic cropping systems.     

鸦胆子褐斑病病原菌鉴定及其防治药剂筛选

通过常规植物病理学和分子生物学方法对人工栽培鸦胆子褐斑病病原菌进行了分离和鉴定,研究了其病原菌对温度和pH的适应性以及对药剂的敏感性。结果表明,引起鸦胆子褐斑病的病原菌为高粱附球菌(Epicoccum sorghinum)。病原菌温度和pH生长曲线回归方程分别为■。由方程计算病原菌最适生长温度为24.6℃,最适pH为5.3。戊唑醇和苯醚甲环唑可作为鸦胆子褐斑病田间防治的备选药剂,且预防效果好于治疗效果。     

Implications of bacterial contaminated seed lots and endophytic colonization by Pseudomonas fuscovaginae on rice establishment

The economic impact of seedborne bacterial diseases on rice production provides a major motivation for research on seed health. This paper reports on the endophytic growth of a rifampicin‐marked strain of the seedborne rice pathogen Pseudomonas fuscovaginae. The bacterium was found in most tested seeds indicating that, even without visible discolouration, seed transmission is possible. Crushed discoloured seeds contained more bacterial cells than did non‐crushed discoloured seeds. These bacteria were released during seed soaking, contaminating clean seed and lowering seed germination. Cells of a rifampicin‐resistant strain of P. fuscovaginae, which had been inoculated onto rice seeds, were subsequently recovered from different growth stages and from different rice tissues, thereby indicating endophytic colonization. These results have implications for seedling establishment, as symptomless seeds do not assure disease‐free seeds, and the presence of seedborne bacteria results in poor germination and poor seedling establishment. Elimination of seedborne bacteria by soaking in sodium hypochlorite can increase seed germination. This could be used in developing control strategies, and, if practised regularly, reduce entry of seedborne disease‐causing organisms into crops, resulting in lower disease pressure.     

Multiphasic analysis of xanthomonads causing bacterial spot disease on tomato and pepper in the Caribbean and central america: evidence for common lineages within and between countries

ABSTRACT Four hundred thirty-three xanthomonad strains isolated from tomato or pepper plants from 32 different fields in four Caribbean and Central American countries were screened for the ability to hydrolyze starch and sodium polypectate and for resistance to copper and streptomycin. Of these, 95 representative strains were further characterized by various phnetic tests, and 63 of these strains were then analyzed by genomic fingerprinting. Most of the strains (>90%) were tolerant to copper. However, there was much more variability in sensitivity to streptomycin. All strains in Guadeloupe and 93% of the strains in Barbados were sensitive to streptomycin. The majority of strains were typical Xanthomonas campestris pv. vesicatoria group A strains. In Barbados, however, a unique group of strains was identified that was serologically similar to group A strains but was amylolytic. These strains were designated A1. The occurrence of X. campestris pv. vesicatoria group B strains in Central America was found to be limited to two fields in Costa Rica and one in Guatemala. No group B strains were identified in the Caribbean, in contrast to common occurrence in the central United States and in South America. T3 strains were not found in this study, despite the recent increase of such strains in Florida and Mexico. Unique strains from Costa Rica belonging to the X. gardneri group were identified. Little linkage was found among phenotypic and rep-polymerase chain reaction (rep-PCR) genomic fingerprinting profiles of the pathogens except at the species/pathovar level; strains displaying virtually identical fingerprint profiles were found to correspond to distinct races and vice versa. The rep-PCR genomic fingerprinting analyses suggest that certain lineages may have evolved or predominated in specific regions or specific countries.     

Rapid detection of Fusarium oxysporum f. sp. lactucae on soil,lettuce seeds and plants using loop‐mediated isothermal amplification

Fusarium oxysporum f. sp. lactucae (FOL) is a soil‐ and seedborne pathogen and the causal agent of fusarium wilt on lettuce. Four races have been identified within FOL, with different worldwide distribution. Several molecular techniques have been used to detect and identify this pathogen; however, not all of them have the optimal characteristics in terms of sensitivity to perform FOL detection in plant and seed material. A loop‐mediated isothermal amplification (LAMP) assay was developed based on the sequence‐characterized amplified region (SCAR) obtained in a previous rapid amplification of polymorphic DNA (RAPD) study. The LAMP assay has been validated according to the EPPO standard PM7/98. The LAMP assay was tested with lettuce seeds, soil and plant material, and can be used successfully to amplify DNA from each of these matrices. In seed lots artificially inoculated with FOL, the detection limit of the LAMP test was 0.004% infected seed.     

Movement of Xanthomonas campestris pv. vitians in the stems of lettuce and seed contamination

Xanthomonas campestris pv. vitians , the causal agent of bacterial leaf spot of lettuce (BLS), can be seedborne, but the mechanism by which the bacteria contaminates and/or infects lettuce seed is not known. In this study, the capacity of X. campestris pv. vitians to enter and translocate within the vascular system of lettuce plants was examined. The stems of 8- to 11-week-old lettuce plants were stab-inoculated, and movement of X. campestris pv. vitians was monitored at various intervals. At 4, 8, 12 and 16 h post-inoculation (hpi), X. campestris pv. vitians was recovered from 2 to 10 cm above (depending on stem length) and 2 cm below the inoculation site. Xanthomonas campestris pv. vitians was also recovered from surface-disinfested stem sections of spray-inoculated plants. Together, these results are consistent with X. campestris pv. vitians invading and moving systemically within the vascular system of lettuce plants. To investigate the mechanism of seed contamination, lettuce plants at the vegetative stage of growth were spray-inoculated with X. campestris pv. vitians and allowed to develop BLS. Seed collected from these plants had a 2% incidence of X. campestris pv. vitians external colonization, but no bacteria were recovered from within the seed.     

Impact of Foliar Diseases on Photosynthesis, Protein Content and Seed Yield of Alfalfa and Efficacy of Fungicide Application

Foliar pathogens attack alfalfa wherever the crop is grown, but their impact, especially on seed production, is poorly understood. In greenhouse trials, leaf spot injury caused by inoculation with various pathogens reduced the crude protein content of infected alfalfa leaves by 22% compared with a healthy control. There was a negative relationship between disease injury and the photosynthetic efficiency of alfalfa plants, as determined by measuring chlorophyll fluorescence in leaves from inoculated vs. non-inoculated seedlings. In field trials at two sites in Alberta from 2001 to 2003, inoculation with Phoma medicaginis increased disease incidence in four of six trials, Phoma sclerotioides increased incidence in four of five trials, and Leptosphaerulina trifolii and Stemphylium botryosum increased incidence in two of six trials. There was a trend for inoculation treatments to reduce seed yield, despite high levels of background infection by indigenous pathogens. The fungicides benomyl and propiconazole inhibited radial growth of Phoma spp. in vitro and reduced disease incidence in inoculated greenhouse experiments. In field trials, applications of benomyl and propiconazole reduced disease incidence, but did not always increase seed yield.     

Effects of maternal light environment on germination and morphological characteristics of Sicyos deppei seeds

The effect was studied of sunlight and far-red (FR) light during seed development, on seed quality and germination of Sicyos deppei G. Don. Seeds exposed to FR during development were lighter in colour and their weight, size and water content were significantly lower. Less than 10% of non-scarified freshly harvested seeds germinated. Scarified, freshly harvested seeds developed under sunlight had a partially negative photoblastic response; both red (R) and FR light inhibited germination. The highest and fastest germination occurred in darkness, probably due to the effect of the high photon flux densities on the phytochrome during seed development. Scarified seeds ripened under FR light, germinated well in FR light and in darkness, but R light inhibited germination. After 6 months of storage, the permeability of S. deppei seeds increased, the partially negative photoblastic response was lost and germination of scarified seeds increased. Specifically, in seeds developed under FR, germination in darkness was faster than for the other light treatments, but slow in darkness for seeds developed under sunlight. The physiological and morphological heteroblastic responses in S. deppei probably extend its seed germination and seedling recruitment periods.     

Evidence for seed transmission and symptomless growth of Ramularia collo‐cygni in barley (Hordeum vulgare)

Ramularia collo‐cygni (Rcc) is becoming an increasing problem for barley growers across Europe. However, the life cycle of the pathogen is only slowly being elucidated. In this study, Rcc DNA was detected in a number of harvested seed samples from 1999 to 2010, with mean levels peaking in winter barley samples in 2009. A number of experiments were carried out to determine whether the pathogen could move from barley seed to seedlings, and also from seed through the developing plant and into the subsequent generation of seed, both in controlled experiments and in field trials. Results from testing of seed indicated that the fungus is widespread at the end of the growing season in harvested grain samples and can be transmitted to developing plants from infected seed stock. Examination of infected seedlings did not reveal the presence of spores but fungal structures were found within the leaf. The location of the fungus within seed was examined, with Rcc DNA found in both embryo and non‐embryo tissue. The implications for barley production of the pathogen being seedborne are discussed.     

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.     

The effect of Phelipanche ramosa infection on the quality of tomato fruit

The influence of the root holoparasite Phelipanche ramosa on the physical characteristics and chemical composition of greenhouse-grown tomato fruits was studied over two growing seasons. The presence of the parasite significantly reduced fruit fresh and dry weight, mesocarp thickness, fruit colour, firmness and titratable acidity. The content of reducing sugars, soluble solids, ash and ascorbic acid was also reduced, but the number of seeds per fruit increased with the parasite. Since some of these traits are important determinants of fruit quality from the consumers' point of view, it is clear that P. ramosa infection reduces the marketable value of tomato. The deterioration in fruit quality worsens over time and hence, by inference, with the intensity of the parasite infection.     

Role of new bacterial surfactins in the antifungal interaction between Bacillus amyloliquefaciens and Fusarium oxysporum

The aim of this work was to investigate the major mechanisms involved in antagonism of BO7, a novel strain of Bacillus amyloliquefaciens isolated from orchard soil, against the vascular wilt fungus Fusarium oxysporum f. sp. lycopersici (Fol). BO7 markedly reduced the incidence of Fol vascular wilt on tomato plants and displayed strong in vitro inhibitory activity against Fol. Scanning electron microscopy demonstrated the ability of BO7 cells to adhere to fungal hyphae and to efficiently colonize tomato roots. The low molecular weight fraction of BO7 culture filtrate displayed high antifungal activity against Fol, resulting in growth inhibition and dramatic alterations of hyphal morphology. Three structurally related surfactin lipopeptides, identified as the major components of the bioactive fraction, were assayed for their inhibitory activity against Fol. One of these compounds exerted a strong and uncommon antifungal activity for lipopeptides of the surfactin family, accounting for most of the inhibitory activity of the BO7 culture filtrate. Among a collection of Fol knockout isolates tested, mutants altered in cell wall structure showed increased sensitivity to the bacterial compounds. These results suggest that the fungal cell wall might have a key role in the sensitivity of Fol towards bacterial surfactins from B. amyloliquefaciens.     

The role of seeds and airborne inoculum in the initiation of leaf blotch (Rhynchosporium secalis) epidemics in winter barley

Both airborne spores of Rhynchosporium secalis and seed infection have been implied as major sources of primary inoculum for barley leaf blotch (scald) epidemics in fields without previous history of barley cropping. However, little is known about their relative importance in the onset of disease. Results from both quantitative real‐time PCR and visual assessments indicated that seed infection was the main source of inoculum in the field trial conducted in this study. Glasshouse studies established that the pathogen can be transmitted from infected seeds into roots, shoots and leaves without causing symptoms. Plants in the field trial remained symptomless for approximately four months before symptoms were observed in the crop. Covering the crop during part of the growing season was shown to prevent pathogen growth, despite the use of infected seed, indicating that changes in the physiological condition of the plant and/or environmental conditions may trigger disease development. However, once the disease appeared in the field it quickly became uniform throughout the cropping area. Only small amounts of R. secalis DNA were measured in 24 h spore‐trap tape samples using PCR. Inoculum levels equivalent to spore concentrations between 30 and 60 spores per m³ of air were only detected on three occasions during the growing season. The temporal pattern and level of detection of R. secalis DNA in spore tape samples indicated that airborne inoculum was limited and most likely represented rain‐splashed conidia rather than putative ascospores.     

Contamination of bean seeds by <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">phaseoli</Emphasis> associated with low bacterial densities in the phyllosphere under field and greenhouse conditions

Xanthomonas axonopodis pv. phaseoli and its variant fuscans are the causal agents of common bacterial blight of bean. Production of seeds is recommended in arid climates with the use of pathogen-free seeds. However, contamination of seeds still occurs in these seed production areas. To verify if low contamination levels of sown seeds could explain these field contaminations, we used seeds that were naturally contaminated with CFBP4834-R, a rifamycin-resistant X. axonopodis pv. phaseoli fuscous strain, to contaminate field plots at different rates. We also inoculated seeds to verify some parameters of plant colonization and seed transmission. In growth chambers, seedling contamination was always successful from seeds contaminated with CFBP4834-R having population sizes greater than 1 × 10³ CFU seed⁻¹ and were not successful below 1 × 10² CFU seed⁻¹. In the greenhouse, the efficiency of contamination of seeds was not significantly different between contaminated plants that had a low or a high CFBP4834-R population size and reached between 40% and 52% whatever the origin of the inoculum (aerial or seed-borne). In field experiments, under low relative humidity, plots with 0.1–0.003% contamination rates or plots sown with seeds that were inoculated with low CFBP4834-R population sizes (1 × 10² and 1 × 10⁴ CFU seed⁻¹) led to an asymptomatic colonization of bean during the entire growing season with low CFBP4834-R population sizes. Seeds were contaminated both in primary and secondary foci. The contamination of seeds without symptom expression during the growing season represents a risk for eventual disease outbreaks.     

Spread and interaction of Pepino mosaic virus (PepMV) and Pythium aphanidermatum in a closed nutrient solution recirculation system: effects on tomato growth and yield

Pepino mosaic virus (PepMV) was shown to be efficiently transmitted between tomato plants grown in a closed recirculating hydroponic system. PepMV was detected in all plant parts after transmission via contaminated nutrient solution using ELISA, immunocapture RT‐PCR, RT‐PCR, electron microscopy, and by inoculation to indicator plants. Detection of PepMV in nutrient solution was only possible after concentration by ultracentrifugation followed by RT‐PCR. Roots tested positive for PepMV 1–3 weeks after inoculation, and subsequently a rapid spread from the roots into the young leaves and developing fruits was found within 1 week. PepMV was only occasionally detected in the older leaves. None of the infected plants showed any symptoms on fruits, leaves or other organs. Pre‐infection of roots of tomato cv. Hildares with Pythium aphanidermatum significantly delayed PepMV root infections. When mechanically inoculated with PepMV at the 2–4 leaf stage, yield loss was observed in all plants. However, only plants of cv. Castle Rock recorded significant yield losses when infected via contaminated nutrient solution. Yield losses induced by infection with PepMV and/or P. aphanidermatum ranged from 0·4 up to 40% depending on experimental conditions.     

Effect of temperature and humidity on the longevity of Orobanche seeds

The effects of moisture and temperature on seed longevity were studied over periods of up to 426 days in three species of Orobanche . Saturated salt solutions were used to achieve equilibrium relative humidities (e.r.h.) between 11% and 85% at temperatures of 20–60 °C. The viability equation describing loss of viability in these conditions applied to the three species such that the lower the temperature and lower the equilibrium relative humidity the greater the longevity. The initial viability differed between species with the seed lot of Orobanche crenata having the lowest viability. However, the relative effect of temperature on longevity and the relative sensitivity of seed longevity to changes in equilibrium relative humidity were the same in all species. Longevity, estimated by the standard deviation of the seed survival curves was greater in Orobanche aegyptiaca and O. crenata than in Orobanche minor , being for example about 100, 103 and 49 days, respectively, at 40 °C, 50% e.r.h. Accurate prediction of longevity is necessary to understand and quantify the population dynamics of weeds and will help the farmer to optimize control measures such as solarization. Taken in conjunction with published data on loss of viability of imbibed seeds, the annual loss of viability on a typical Eritrean farm is predicted to be about 38%.     

Effects of inoculum density,leaf wetness duration and nitrate concentration on the occurrence of lettuce leaf spot

In Ehime Prefecture, Japan, lettuce leaf spot (Septoria lactucae) caused huge losses in marketable lettuce yields. To explore potential measures to control disease outbreaks, the effects of inoculum density, leaf wetness duration and nitrate concentration on the development of leaf spot on lettuce (Lactuca sativa) were evaluated. Conidia were collected from diseased plants in an infested field by single-spore isolation and were used to inoculate potted lettuce plants with different conidial concentrations. Lesions developed on inoculated lettuce plants at inoculum concentrations from 10⁰ to 10⁶ conidia/ml. The disease was more severe when the inoculum exceeded 10² conidia/ml, and severity increased with increasing concentrations. Assessment of the relationship between disease development and the duration of postinoculation leaf wetness revealed that symptoms appeared when the inoculated plants remained wet for 12 h or longer. The number of lesions and total nitrogen content in the lettuce leaves both increased when nitrate was applied.