SSR assessment of Phytophthora infestans populations on tomato and potato in British gardens demonstrates high diversity but no evidence for host specialization

Phytophthora infestans populations can differ in composition as a result of host specialization on tomato and potato hosts. In Great Britain many amateur gardeners grow outdoor tomatoes but there is little or no commercial tomato production outdoors. This study analysed isolates of P. infestans from British gardens with 12 multiplexed simple sequence repeat markers that are used to monitor the disease on commercial potato crops. Samples of P. infestans from tomato hosts were collected in 3 years and from potato in 1 year from across Great Britain. Seven previously unreported clonal lineages were detected in garden populations and higher frequencies of unique clonal lineages (28–40%) were present compared with populations from British commercial potato crops reported elsewhere. Garden populations had a lower proportion (11–48% less) of the most common lineages (13_A2 and 6_A1) that together made up at least 86% of the commercial potato populations during the sampling period. Host species accounted for only 2·0% of molecular variance detected between garden potato‐ and tomato‐hosted samples. No significant difference in clonal lineage composition was found between host species in Great Britain and this could be due to the whole P. infestans population overwintering on potato. British garden populations on both hosts were much more diverse than those on commercial potato crops; this finding may be influenced by less frequent fungicide use by gardeners and a higher diversity of unsprayed susceptible potato cultivars, enabling metalaxyl‐sensitive and less aggressive genotypes to survive in gardens.     

Resistance against Botrytis cinerea in smooth leaf pruning wounds of tomato does not depend on major disease signalling pathways

In high‐tech, heated tomato glasshouses, stem infections caused by Botrytis cinerea usually end up girdling the stem, resulting in plant death and consequently high economic losses. Such infections originate primarily from wounds created during leaf pruning, a common cultural practice in which it is intended to remove leaves completely, resulting in smooth stem wounds. However, hasty leaf pruning often results in numerous petiole stubs accidentally left behind. In this study analysis of disease incidences clearly proved that pruning leaves flush to the stem resulted in absolute resistance of the stem wounds, whereas petiole stubs displayed a high level of susceptibility to B. cinerea. Postponing inoculation of wounds after pruning indicated that development of nearly complete resistance occurs within 48 h after deleafing. Monitoring of the wound wetness period showed that drying of the wound surface is not the cause of the decreased susceptibility, contrary to what was commonly believed. Tomato mutants deficient in disease signalling showed altered phenotypes for susceptibility to B. cinerea, indicating that defences against this pathogen in petiole stubs depend on ethylene signalling. Additionally, the decreased susceptibility of mutants deficient in the biosynthesis of jasmonates and abscisic acid suggest an antagonistic effect of these signal molecules. On the other hand, resistance of smooth stem wounds could not be altered by disruption of salicylic acid, ethylene, jasmonate or abscisic acid signalling. This indicates that this remarkable absolute resistance to B. cinerea does not depend on the major disease signalling pathways.     

Colonization of lettuce cultivars and rotation crops by Fusarium oxysporum f. sp. lactucae,the cause of fusarium wilt of lettuce

The severity of fusarium wilt is affected by inoculum density in soil, which is expected to decline during intervals when a non‐susceptible crop is grown. However, the anticipated benefits of crop rotation may not be realized if the pathogen can colonize and produce inoculum on a resistant cultivar or rotation crop. The present study documented colonization of roots of broccoli, cauliflower and spinach by Fusarium oxysporum f. sp. lactucae, the cause of fusarium wilt of lettuce. The frequency of infection was significantly lower on all three rotation crops than on a susceptible lettuce cultivar, and the pathogen was restricted to the cortex of roots of broccoli. However, F. oxysporum f. sp. lactucae was isolated from the root vascular stele of 7·4% of cauliflower plants and 50% of spinach plants that were sampled, indicating a greater potential for colonization and production of inoculum on these crops. The pathogen was also recovered from the root vascular stele of five fusarium wilt‐resistant lettuce cultivars. Thus, disease‐resistant plants may support growth of the pathogen and thereby contribute to an increase in soil inoculum density. Cultivars that were indistinguishable based on above‐ground symptoms, differed significantly in the extent to which they were colonized by F. oxysporum f. sp. lactucae. Less extensively colonized cultivars may prove to be superior sources of resistance to fusarium wilt for use in breeding programmes.     

Detection of Tomato yellow leaf curl virus in imported tomato fruit in northern Europe

Imported tomato fruits infected with Tomato yellow leaf curl virus (TYLCV) were identified on the market in northern Europe using paper‐based FTA Classic Cards (Whatman), polymerase chain reaction (PCR) and partial DNA sequence analysis. Trade tomatoes originating from southern Europe, Africa and the Middle East were sampled in Estonia and Sweden, and tested for infection with begomoviruses. Out of 100 batches analysed with five fruits sampled in each batch (58 batches from Estonia and 42 from Sweden), 20 batches were positive (16 from Estonia and four from Sweden). Rolling circle amplification (RCA) and full‐length genome sequence analysis of one isolate collected in Estonia and one isolate in Sweden, revealed highest nucleotide sequence identity at 99% to TYLCV‐IL for the Estonian isolate and at 97% to TYLCV‐Mld for the Swedish isolate. In this study, TYLCV was identified for the first time in imported tomato fruits on the market in northern Europe. FTA cards proved to be an effective means to collect, extract and store begomovirus DNA from tomato fruits and the subsequent molecular analysis.     

Effects of plant age on disease development and virulence of Clavibacter michiganensis subsp. michiganensis on tomato

The effect of plant age at the time of inoculation on the severity of bacterial wilt and canker disease caused by Clavibacter michiganensis subsp. michiganensis (Cmm) was examined in six greenhouse experiments. The period during which inoculations led to wilt and death of tomato plants was defined. This period, designated ‘window of vulnerability’, ranged from transplanting to the 17‐ to 18‐leaf stage. Plants inoculated after this period expressed disease symptoms but did not wilt or die. No significant changes in disease incidence were observed when leaves of different ages were inoculated. Yield accumulation was significantly reduced in plants inoculated within the window of vulnerability compared with those inoculated after this period. Expression of virulence genes, viz. celA, encoding a secreted cellulase, and the serine protease‐encoding pat‐1, chpC and ppaA, was induced during the early stages after inoculation in plants inoculated within the window of vulnerability. Differences in Cmm population between plants inoculated within and outside of this period were insignificant after the first week post‐inoculation, indicating that differences in disease severity, yield loss and expression of virulence determinants are not correlated with Cmm population level. Results suggest that implementation of precautionary measures during the window of vulnerability to avoid secondary spread of Cmm will have a season‐long effect on plant mortality and may minimize, or even prevent, yield losses.     

A new race of Fusarium oxysporum f. sp. lactucae of lettuce

Fusarium oxysporum f. sp. lactucae, the causal agent of fusarium wilt of lettuce (Lactuca sativa), occurs in most countries in which lettuce is grown and causes serious economic losses. Three races (1, 2 and 3) of the pathogen have previously been identified on the basis of their ability to cause disease on differential lettuce cultivars, as well as by means of molecular tools developed to characterize different races of this pathogen. Only race 1 has been detected in Europe so far. In this study, two isolates of F. oxysporum, obtained from lettuce plants grown in the Netherlands showing symptoms of wilt, have been characterized by combining the study of pathogenicity with differential cultivars of lettuce and molecular assays to determine whether the isolates are different from the known races of F. oxysporum f. sp. lactucae. This study reports the presence of F. oxysporum f. sp. lactucae for the first time in the Netherlands. The causal pathogen has been identified, using the IRAP‐SCAR technique, as a new race of F. oxysporum f. sp. lactucae. Specific primers have been designed to identify this new race.     

High‐resolution melting analysis for rapid detection and characterization of Botrytis cinerea phenotypes resistant to fenhexamid and boscalid

A novel, high‐resolution melting (HRM) analysis was developed to detect single nucleotide polymorphisms (SNPs) associated with resistance to fenhexamid (hydroxyanilides) and boscalid (succinate dehydrogenase inhibitors) in Botrytis cinerea isolates. Thirty‐six single‐spore isolates arising from 13 phenotypes were selected and tested for fungicide sensitivity. Germ tube elongation assays showed two distinct sensitivity levels for each fungicide. Sequencing revealed that resistance to fenhexamid was due to a nucleotide change in the erg27 gene, resulting in an amino acid replacement of phenylalanine (F) with serine (S) or valine (V) at position 412 of the protein, whereas in isolates resistant to boscalid, a nucleotide change in the sdhB gene resulted in the replacement of histidine (H) with arginine (R) or tyrosine (Y) at position 272 of the respective protein. In each case, melting curve analysis generated three distinct profiles corresponding to the presence of each nucleotide in the targeted areas. HRM analysis successfully detected and differentiated the substitutions associated with resistance to both fungicides. In vitro bioassays, direct sequencing and high‐resolution melting analysis showed a 100% correlation with detection of resistance. The results demonstrate the utility of HRM analysis as a potential molecular tool for routine detection of fungicide resistance using known polymorphic genes of B. cinerea populations.     

Molecular and biological characterization of two potyviruses infecting lettuce in southeastern France

Several potyviruses affect lettuce (Lactuca sativa) and chicory (Cichorium spp.) crops worldwide and are important constraints for production because of the direct losses that they induce and/or because of their seed transmission. Here, the molecular and biological properties are described of two potyviruses that were recently isolated from lettuce plants showing mosaic or strong necrotic symptoms in an experimental field in southeastern France. The first potyvirus belongs to the species Endive necrotic mosaic virus and is present in a large number of wild plant species, especially Tragopogon pratensis. It is unable to infect lettuce cultivars with a resistance to Turnip mosaic virus that is present in many European cultivars and probably conferred by the Tu gene. The second potyvirus belongs to the tentative species lettuce Italian necrotic virus and was not observed in wild plants. It infected all tested lettuce cultivars. Wild accessions of Lactuca serriola, Lactuca saligna, Lactuca virosa and Lactuca perennis were identified as resistant to one or the other potyvirus and could be used for resistance breeding in lettuce. No resistance against these two potyviruses was observed in the tested Cichorium endivia cultivars. In contrast, all tested Cichorium intybus cultivars or accessions were resistant.     

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.     

Evaluation and QTL mapping of resistance to powdery mildew in lettuce

Lettuce (Lactuca sativa) is the major leafy vegetable that is susceptible to powdery mildew disease under greenhouse and field conditions. Quantitative trait loci (QTLs) for resistance to powdery mildew under greenhouse conditions were mapped in an interspecific population derived from a cross between susceptible L. sativa cultivar Salinas and the highly susceptible L. serriola accession UC96US23. Four significant QTLs were detected on linkage groups LG 1 (pm‐1.1), LG 2 (pm‐2.1 and pm‐2.2) and LG 7 (pm‐7.1), each explaining between 35 to 42% of the phenotypic variation. The four QTLs are not located in the documented hotspots of lettuce resistance genes. Alleles for the disease resistance at the four QTLs originated from both parents (two from each), demonstrating that even highly susceptible accessions may provide alleles for resistance to powdery mildew. These QTLs appeared to operate during limited periods of time. Results of the field trials with F_2:3 and F_3:4 families derived from a Soraya (moderately resistant) × Salinas cross demonstrated effective transfer of resistance to powdery mildew in this material. An integrated rating approach was used to estimate relative levels of resistance in 80 cultivars and accessions tested in a total of 23 field and greenhouse experiments. Generally, very low resistance was observed in crisphead‐type lettuces, while the highest relative resistance was recorded in leaf and butterhead types. Comparison of two disease assessment methods (percentage rating and the area under the disease progress steps, AUDPS) for detection of QTLs shows that the two approaches complement each other.     

Characterization and pathogenicity assessment of Plectosphaerella species associated with stunting disease on tomato and pepper crops in Italy

This study follows a survey carried out in 2012 and 2013 on tomato and pepper crops in the Foggia province (southern Italy), for morphological, molecular and pathogenicity analyses of Plectosphaerella fungi. The Plectosphaerella genus includes several species that are pathogens of horticultural plants. The survey identified tomato and pepper crops that showed abundant wilt, leaf yellowing, and discolouration and necrosis of roots, plus collar and stem symptoms. Different fungi including Plectosphaerella spp. were isolated from tissues with and without symptoms. Subsequent molecular and morphological studies identified first records of P. citrulli infecting tomato and pepper, and P. pauciseptata and P. ramiseptata infecting pepper. Pathogenicity testing confirmed that most isolated species of Plectosphaerella caused symptoms on tomato and pepper, with P. ramiseptata the most aggressive. On the basis of these data, it is considered that Plectosphaerella species may cause stunting disease in tomato and pepper.     

Development,characterization and application of genomic SSR markers for the oat stem rust pathogen Puccinia graminis f. sp. avenae

Oat stem rust, caused by Puccinia graminis f. sp. avenae (Pga), is one of the most severe diseases of oats worldwide. Population studies are scarce for this pathogen, mainly due to the lack of polymorphic molecular markers suitable for genetic analysis. In this study, an Australian Pga isolate was sequenced, the abundance of simple sequence repeats (SSRs) was determined and PCR‐based polymorphic markers suitable for genetic diversity analysis were developed. The amplification of 194 primer pairs was initially assessed using a set of 12 isolates of different cereal rust species and their formae speciales. A high frequency of cross‐species amplification was observed for most markers; however, 36 SSRs were diagnostic for P. graminis only. A subset of 19 genome‐derived SSRs were deemed useful for genetic diversity analysis of Pga and were assessed on 66 Pga isolates from Australia, Brazil and Sweden. Brazilian and Australian isolates were characterized by one and two predominant clonal lineages, respectively. In contrast, the Swedish isolates, previously shown to undergo sexual recombination, were highly diverse (nine distinct genotypes out of 10 isolates) and divided into two subpopulations. The genome‐derived SSR markers developed in this study were well suited to the population studies undertaken, and have diagnostic capabilities that should aid in the identification of unknown rust pathogen species.     

Genetic population structure and fungicide resistance of Botrytis cinerea in pear orchards in the Western Cape of South Africa

Botrytis cinerea isolates from pear blossoms (Pyrus communis) in South Africa were collected from four orchards in two production areas in the Western Cape. The cryptic species status based on vegetative‐incompatibility alleles of the Bc‐hch gene indicated that all the isolates belonged to B. cinerea. A microsatellite analysis of B. cinerea populations was performed to assess the genetic population structure. Total gene diversity (H) was high, with a mean of 0.69 across all populations. Some genotype flow was evident between orchards as indicated by the spread of microsatellite multilocus genotypes, in agreement with the moderate, but significant population differentiation among orchards (mean φ_PT = 0.118, P = 0.001). Index of association analyses (I_A and r?_d) suggest that the populations reproduce mostly asexually, even though mating type distribution did not differ significantly from a 1:1 ratio, suggesting frequency‐dependent selection. Isolates resistant to benomyl were evident in one orchard only. This orchard was also significantly differentiated from all other populations, suggesting infrequent localized selection for benomyl resistance. Overall, the findings of this study highlight the dangers of a mixed reproduction system, and stress the importance of regularly monitoring fungicide resistance levels towards developing more efficient management practices.     

Long‐lasting β‐aminobutyric acid‐induced resistance protects tomato fruit against Botrytis cinerea

Minimizing losses to pests and diseases is essential for producing sufficient food to feed the world's rapidly growing population. The necrotrophic fungus Botrytis cinerea triggers devastating pre‐ and post‐harvest yield losses in tomato (Solanum lycopersicum). Current control methods are based on the pre‐harvest use of fungicides, which are limited by strict legislation. This investigation tested whether induction of resistance by β‐aminobutyric acid (BABA) at different developmental stages provides an alternative strategy to protect post‐harvest tomato fruit against B. cinerea. Soil‐drenching plants with BABA once fruit had already formed had no impact on tomato susceptibility to B. cinerea. However, BABA application to seedlings significantly reduced post‐harvest infection of fruit. This resistance response was not associated with a yield reduction; however, there was a delay in fruit ripening. Untargeted metabolomics revealed differences between fruit from water‐ and BABA‐treated plants, demonstrating that BABA triggered a defence‐associated metabolomics profile that was long lasting. Targeted analysis of defence hormones suggested a role of abscisic acid (ABA) in the resistance phenotype. Post‐harvest application of ABA to the fruit of water‐treated plants induced susceptibility to B. cinerea. This phenotype was absent from the ABA‐exposed fruit of BABA‐treated plants, suggesting a complex role of ABA in BABA‐induced resistance. A final targeted metabolomic analysis detected trace residues of BABA accumulated in the red fruit. Overall, it was demonstrated that BABA induces post‐harvest resistance in tomato fruit against B. cinerea with no penalties in yield.     

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.     

Lack of host specificity of Colletotrichum spp. isolates associated with anthracnose symptoms on mango in Brazil

The aim of the present study was to analyse the genetic and pathogenic variability of Colletotrichum spp. isolates from various organs and cultivars of mango with anthracnose symptoms, collected from different municipalities of São Paulo State, Brazil. Colletotrichum gloeosporioides isolates from symptomless citrus leaves and C. acutatum isolates from citrus flowers with post‐bloom fruit drop symptoms were included as controls. Sequencing of the ITS region allowed the identification of 183 C. gloeosporioides isolates from mango; only one isolate was identified as C. acutatum. amova analysis of ITS sequences showed larger genetic variability among isolates from the same municipality than among those from different populations. fAFLP markers indicated high levels of genetic variability among the C. gloeosporioides isolates from mango and no correlation between genetic variability and isolate source. Only one C. gloeosporioides mango isolate had the same genotype as the C. gloeosporioides isolates from citrus leaves, as determined by ITS sequencing and fAFLP analysis. Pathogenicity tests revealed that C. gloeosporioides and C. acutatum isolates from either mango or citrus can cause anthracnose symptoms on leaves of mango cvs Palmer and Tommy Atkins and blossom blight symptoms in citrus flowers. These outcomes indicate a lack of host specificity of the Colletotrichum species and suggest the possibility of host migration.     

Disease development and genotypic diversity of Puccinia graminis f. sp. avenae in Swedish oat fields

The disease development and population structure of Puccinia graminis f. sp. avenae, which causes stem rust on oats, were studied to investigate if sexual reproduction plays an important role in the epidemiology of the disease. The genetic population structure of P. graminis f. sp. avenae in Sweden was investigated by sampling 10 oat fields in July and August 2008 and seven fields during the same period in 2009. Nine single‐pustule isolates were first used to test simple sequence repeat (SSR) markers developed for P. graminis f. sp. tritici. Eleven of the 68 tested SSR markers were useful for genotyping P. graminis f. sp. avenae. For the main study, DNA from single uredinia was extracted and the SSR markers were used to genotype 472 samples. Both allelic and genotypic diversity were high in all fields, indicating that P. graminis f. sp. avenae undergoes regular sexual reproduction in Sweden. No significant relationship between genetic and geographic distances was found. Disease development was studied on two farms during 2008 and 2009. The apparent infection rates ranged between 0·17 and 0·55, indicating the potential for rapid disease development within fields. The incidence of oat stem rust has increased recently in Sweden. One possible explanation is a resurgence of its alternate host, barberry (Berberis spp.), after the repeal of the barberry eradication law in 1994. Barberry is present in several grain‐producing areas in Sweden, which supports the conclusion that P. graminis f. sp. avenae undergoes regular sexual reproduction there.     

Effect of different organic amendments on lettuce fusarium wilt and on selected soilborne microorganisms

This study evaluated the effect of different organic amendments on lettuce fusarium wilt caused by Fusarium oxysporum f. sp. lactucae in pots under controlled conditions. Their effects on the density of the pathogen, on the total fungi and on fluorescent Pseudomonas spp. were also evaluated after two subsequent lettuce crops. A significant reduction in the severity of the symptoms of F. oxysporum f. sp. lactucae was found after the use of Brassica carinata pellets (52–79% reduction) and compost (49–67% reduction), while Brassica green manure and cattle and chicken manure only provided partial control of fusarium wilt. However, variations in effectiveness were observed for the same treatment in repeated trials. In general, an increase was observed in Pseudomonas and a decrease in fungal populations in the growing medium, which was obtained by mixing a blonde sphagnum peat and a sandy loam soil with B. carinata pellets and compost after two consecutive cropping cycles. Prolonging the Brassica and compost treatments from 30 to 60 days did not significantly affect disease severity, plant growth or the microbial population of the total fungi or Pseudomonas. The largest lettuce biomass was obtained in the non‐inoculated growing medium amended with brassica flour, chicken manure, B. carinata pellets and compost, as a consequence of fertilization. The treatment with B. juncea green manure, B. carinata (pellets and flour) and compost applied 30 days before planting led to promising results and merits further investigation for use under field conditions.