Virulence of ‘Dickeya solani’ and Dickeya dianthicola biovar‐1 and ‐7 strains on potato (Solanum tuberosum)

Studies were conducted to explain the relative success of ‘Dickeya solani’, a genetic clade of Dickeya biovar 3 and a blackleg‐causing organism that, after recent introduction, has spread rapidly in seed potato production in Europe to the extent that it is now more frequently detected than D. dianthicola. In vitro experiments showed that both species were motile, had comparable siderophore production and pectinolytic activity, and that there was no antagonism between them when growing. Both ‘D. solani’ and biovar 1 and biovar 7 of D. dianthicola rotted tuber tissue when inoculated at a low density of 10³ CFU mL^?1. In an agar overlay assay, D. dianthicola was susceptible to 80% of saprophytic bacteria isolated from tuber extracts, whereas ‘D. solani’ was susceptible to only 31%, suggesting that ‘D. solani’ could be a stronger competitor in the potato ecosystem. In greenhouse experiments at high temperatures (28°C), roots were more rapidly colonized by ‘D. solani’ than by biovar 1 or 7 of D. dianthicola and at 30 days after inoculation higher densities of ‘D. solani’ were found in stolons and progeny tubers. In co‐inoculated plants, fluorescent protein (GFP or DsRed)‐tagged ‘D. solani’ outcompeted D. dianthicola in plants grown from vacuum‐infiltrated tubers. In 3 years of field studies in the Netherlands with D. dianthicola and ‘D. solani’, disease incidence varied greatly annually and with strain. In summary, ‘D. solani’ possesses features which allow more efficient plant colonization than D. dianthicola at high temperatures. In temperate climates, however, tuber infections with ‘D. solani’ will not necessarily result in a higher disease incidence than infections with D. dianthicola, but latent seed infection could be more prevalent.     

Characterization and epidemiology of Colletotrichum acutatum sensu lato (C. chrysanthemi) causing Carthamus tinctorius anthracnose

In 2012, Colletotrichum isolates were collected from field‐grown safflower (Carthamus tinctorius) crops in central Italy from plants exhibiting typical anthracnose symptoms. Colletotrichum isolates were also collected from seed surfaces and from within seeds. The genetic variability of these isolates was assessed by a multilocus sequencing approach and compared with those from Colletotrichum chrysanthemi and Colletotrichum carthami isolates from different geographic areas and other Colletotrichum acutatum sensu lato‐related isolates. Phylogenetic analysis revealed that all of the strains isolated from C. tinctorius belonged to the species described as C. chrysanthemi, whereas all of the strains belonging to C. carthami had been isolated from Calendula officinalis. Phenotypic characterization of isolates was performed by assessing growth rates at different temperatures, morphology of colonies on potato dextrose agar (PDA) and the size of conidia. All C. chrysanthemi isolates from safflower had similar growth rates at different temperatures, comparable colony morphologies when grown on PDA and conidial sizes consistent with previously described C. chrysanthemi isolates. Pathogenicity tests were performed by artificially inoculating both seeds and plants and confirmed the seedborne nature of this pathogen. When inoculated on plants, C. chrysanthemi caused the typical symptoms of anthracnose on leaves. This is the first record of this pathogen on C. tinctorius in Italy, and it presents an updated characterization of Colletotrichum isolates pathogenic to safflowers in Europe.     

Inoculation of Malus genotypes with a set of Erwinia amylovora strains indicates a gene‐for‐gene relationship between the effector gene eop1 and both Malus floribunda 821 and Malus ‘Evereste’

The Gram‐negative bacterium Erwinia amylovora, causal agent of fire blight disease in pome fruit trees, encodes a type three secretion system (T3SS) that translocates effector proteins into plant cells that collectively function to suppress host defences and enable pathogenesis. Until now, there has only been limited knowledge about the interaction of effector proteins and host resistance presented in several wild Malus species. This study tested disease responses in several Malus wild species with a set of effector deletion mutant strains and several highly virulent E. amylovora strains, which are assumed to influence the host resistance response of fire blight‐resistant Malus species. The findings confirm earlier studies that deletion of the T3SS abolished virulence of the pathogen. Furthermore, a new gene‐for‐gene relationship was established between the effector protein Eop1 and the fire blight resistant ornamental apple cultivar Evereste and the wild species Malus floribunda 821. The results presented here provide new insights into the host–pathogen interactions between Malus sp. and E. amylovora.     

Disease protection and growth promotion of potatoes (Solanum tuberosum L.) by Paenibacillus dendritiformis

One of the challenges in developing plant‐beneficial bacterial agents for agricultural application is ensuring that an effective selection and screening procedure is in place. The sporadic success of using bacterial agents in the field is usually due to the inability of added bacteria to compete with the local microorganisms. In the present study, the effectiveness of Paenibacillus dendritiformis, a unique pattern‐forming, Gram‐positive, soil bacterium, to reduce disease indices and increase yield in potato crops was examined. This bacterium was chosen as a potential agent based on genome analysis carried out in previous studies. In vitro laboratory experiments, as well as three greenhouse and one field experiment, were conducted. The results show that, in agreement with the hypothesis, P. dendritiformis significantly reduced the maceration area of tuber slices infected by Pectobacterium carotovorum subsp. carotovorum, significantly reduced disease indices in greenhouse experiments and significantly increased tuber yield of infected plants in the field. This work demonstrates the potential of preliminary screening based on genome analysis to identify effective biocontrol agents.     

Most Colletotrichum species associated with tree tomato (Solanum betaceum) and mango (Mangifera indica) crops are not host‐specific

An important constraint for crop production in Colombia is the high incidence of anthracnose caused by Colletotrichum species. Although several studies have focused on these fungi, the relationship between the different fungal species within the genus and their hosts and whether they display any host preference or host specificity has yet to be examined. In Colombia, diseases caused by Colletotrichum species are particularly severe in mango (Mangifera indica) and tree tomato (Solanum betaceum) crops. In a previous investigation, the Colletotrichum phylogenetic species attacking these crops were identified. The present study aimed to determine whether isolates collected from tree tomato and mango showed host preference or host specificity by assessing aggressiveness, spore density, latent period, and fitness of each strain on the two hosts. In the departments of Cundinamarca and Tolima, Colombia, isolates were collected from plants that presented typical anthracnose symptoms and were identified as C. acutatum, C. asianum, C. boninense, C. gloeosporioides, C. tamarilloi and C. theobromicola. Inoculation of conidia of each isolate onto both hosts showed isolates had no host preference and only the C. gloeosporioides isolate showed host specificity. However, in general, isolates produced a higher spore density when inoculated on the alternate host, which may indicate a difference in the degree of adaptation to each host. Statistical analyses of the assessed parameter values revealed that isolates use different infection strategies when infecting each host. In light of these results, the implications of using quantitative estimations of fitness when studying fungal pathogens are discussed.     

Studies on the interaction between the biocontrol agent,Serratia plymuthica A30, and blackleg‐causing Dickeya sp. (biovar 3) in potato (Solanum tuberosum)

Interactions between Serratia plymuthica A30 and a blackleg‐causing biovar 3 Dickeya sp. were examined. In a potato slice assay, S. plymuthica A30 inhibited tissue maceration caused by Dickeya sp. IPO2222 when co‐inoculated at a density at least 10 times greater than that of the pathogen. In glasshouse experiments, population dynamics of the antagonist and of the pathogen in planta were studied by dilution plating and confocal laser scanning microscopy (CLSM) using fluorescent protein‐tagged strains. Pathogen‐free minitubers were vacuum‐infiltrated with DsRed‐tagged Dickeya sp. IPO2222 and superficially treated during planting with a water suspension containing GFP‐tagged S. plymuthica A30. A30 reduced the blackleg incidence from 55% to 0%. Both the pathogen and the antagonist colonized the seed potato tubers internally within 1 day post‐inoculation (dpi). Between 1 and 7 dpi, the population of A30 in tubers increased from 10¹ to c. 10³ CFU g^?1 and subsequently remained stable until the end of the experiment (28 dpi). Populations of A30 in stems and roots increased from c. 10² to c. 10⁴ CFU g^?1 between 7 and 28 dpi. Dilution plating and CLSM studies showed that A30 decreased the density of Dickeya sp. populations in plants. Dilution plating combined with microscopy allowed the enumeration of strain A30 and its visualization in the vascular tissues of stem and roots and in the pith of roots, as well as its adherence to and colonization of the root surface. The implications of these finding for the use of S. plymuthica A30 as a biocontrol agent are discussed.     

Morphological and molecular diversity of Colletotrichum spp. causing pepper spot and anthracnose of lychee (Litchi chinensis) in Australia

Since the 1980s a new disease has been affecting Australian lychee. Pepper spot appears as small, black superficial lesions on fruit, leaves, petioles and pedicels and is caused by Colletotrichum gloeosporioides, the same fungus that causes postharvest anthracnose of lychee fruit. The aim of this study was to determine if a new genotype of C. gloeosporioides is responsible for the pepper spot symptom. Morphological assessments, arbitrarily‐primed PCR (ap‐PCR) and DNA sequencing studies did not differentiate isolates of C. gloeosporioides from anthracnose and pepper spot lesions. The ap‐PCR identified 21 different genotypes of C. gloeosporioides, three of which were predominant. A specific genotype identified using ap‐PCR was associated with the production of the teleomorph in culture. Analysis of sequence data of ITS and β‐tubulin regions of representative isolates did not group the lychee isolates into a monophyletic clade; however, given the majority of the isolates were from one of three genotypes found using ap‐PCR, the possibility of a lychee specific group of C. gloeosporioides is discussed.     

Characterization of Pseudomonas syringae pv. actinidiae (Psa) isolated from France and assignment of Psa biovar 4 to a de novo pathovar: Pseudomonas syringae pv. actinidifoliorum pv. nov.

Since 2008, bacterial canker of kiwifruit (Actinidia deliciosa and A. chinensis) caused by Pseudomonas syringae pv. actinidiae (Psa) has resulted in severe economic losses worldwide. Four biovars of Psa can be distinguished based on their biochemical, pathogenicity and molecular characteristics. Using a range of biochemical, molecular and pathogenicity assays, strains collected in France since the beginning of the outbreak in 2010 were found to be genotypically and phenotypically diverse, and to belong to biovar 3 or biovar 4. This is the first time that strains of biovar 4 have been isolated outside New Zealand or Australia. A multilocus sequence analysis based on four housekeeping genes (gapA, gltA, gyrB and rpoD) was performed on 72 strains representative of the French outbreak. All the strains fell into two phylogenetic groups: one clonal corresponding to biovar 3, and the other corresponding to biovar 4. This second phylogenetic group was polymorphic and could be divided into four lineages. A clonal genealogy performed with a coalescent approach did not reveal any common ancestor for the 72 Psa strains. Strains of biovar 4 are substantially different from those of the other biovars: they are less aggressive and cause only leaf spots whereas Psa biovars 1, 2 and 3 also cause canker and shoot die‐back. Because of these pathogenic differences, which were supported by phenotypic, genetic and phylogenetic differences, it is proposed that Psa biovar 4 be renamed Pseudomonas syringae pv. actinidifoliorum pv. nov. Strain CFBP 8039 is designated as the pathotype strain.     

Transmission of ‘Candidatus Phytoplasma pyri’ by naturally infected Cacopsylla pyri to peach,an approach to the epidemiology of peach yellow leaf roll (PYLR) in Spain

Peach orchards in the northeast of Spain were severely affected in 2012 by a previously unreported disease in this area. The symptoms included early reddening, leaf curling, decline, abnormal fruits, and in some cases death of the peach trees. All the infected peach samples were positive for ‘Candidatus Phytoplasma pyri’, but none were infected by the ‘Ca. Phytoplasma prunorum’. In this work, potential vectors able to transmit ‘Ca. Phytoplasma pyri’ from pear to peach and between peach trees were studied and their infective potential was analysed at different times of the year. Transmission trials of the phytoplasma with potential vectors to an artificial feeding medium for insects and to healthy peach trees were conducted. Additionally, isolated phytoplasmas were genetically characterized to determine which isolates were able to infect peach trees. Results showed that the only insect species captured inside peach plots that was a carrier of the ‘Ca. Phytoplasma pyri’ phytoplasma was Cacopsylla pyri. Other insect species captured and known to be phytoplasma transmitters were present in very low numbers, and were not infected with ‘Ca. Phytoplasma pyri’ phytoplasma. A total of 1928 individuals of C. pyri were captured in the peach orchards, of which around 49% were phytoplasma carriers. All the peach trees exposed to C. pyri in 2014, and 65% in 2015, were infected by ‘Ca. Phytoplasma pyri’ 1 year after exposure, showing that this species is able to transmit the phytoplasma to peach. Molecular characterization showed that some genotypes are preferentially determined in peach.     

A rapid virulence assay for the Dutch elm disease fungus Ophiostoma novo‐ulmi by inoculation of apple (Malus × domestica ‘Golden Delicious’) fruits

Large‐scale virulence tests using trees or saplings are expensive, time‐consuming and require a considerable amount of space. The suitability of using ‘Golden Delicious’ apples as a rapid screen for identifying Ophiostoma novo‐ulmi transformants with reduced virulence was thus evaluated. When a collection of O. novo‐ulmi field isolates belonging to subspecies novo‐ulmi or americana was inoculated to apples, members of subsp. novo‐ulmi induced, on average, larger necrotic lesions than subsp. americana isolates. The size of the lesions on apples was not correlated with mycelial growth rate of isolates on nutrient agar. Insertional mutants from O. novo‐ulmi subsp. novo‐ulmi isolate H327 were inoculated to ‘Golden Delicious’ apples and Ulmus parvifolia × U. americana saplings in parallel experiments. Results clearly indicated that the O. novo‐ulmi transformants included several exhibiting significantly altered levels of virulence. Variability among replicates within a treatment was reduced in apple inoculation data compared to elm sapling data. Overall, the ‘Golden Delicious’ apple assay was found to be an excellent means for rapidly assessing the virulence level of O. novo‐ulmi isolates.     

Methodology of virulence screening and race characterization of Plasmopara halstedii,and resistance evaluation in sunflower – a review

Sunflower downy mildew is a disease of high global economic impact as well as a causal agent that is extremely difficult to eradicate. During the past decades, several approaches for the determination of Plasmopara halstedii (Ph) races have been used worldwide and are discussed in this review. Procedures of isolation, cultivation and maintenance of Ph isolates, as well as the screening of sunflower for resistance, are also critically reviewed. The predominant, globally used resistance screening protocol is a ‘whole seedling immersion’ inoculation. ‘Soil drench’ inoculation allows more precise control of the number of Ph zoosporangia applied to a single sunflower seedling. A detached leaf assay has been described, but it has been used mainly for Ph subcultivation and fungicide tests. For race determination, a differential set consisting of nine sunflower genotypes has been used since 1988, coupled with a numerical triplet code for virulence phenotyping of Ph. The increasing variability in global Ph populations has demonstrated the inadequacy of the current set of differentials, and several researchers have proposed additional public lines as new differentials. Furthermore, bulk isolates may show different results in repeated tests, as Ph may contain genetically distinct zoospores within a single zoosporangium. For precise race determination, single zoosporangia or single zoospore isolates are advisable. However, due to low success of isolation, approximately 1–2%, this method cannot be applied in routine Ph race screening. Methods surveyed in this review have a broad spectrum of applications, including taxonomic studies.     

Incidence and distribution of ‘Candidatus Phytoplasma prunorum’ and its vector Cacopsylla pruni in Spain: an approach to the epidemiology of the disease and the role of wild Prunus

‘Candidatus Phytoplasma prunorum’ is the causal agent of the European stone fruit yellows (ESFY) disease. This phytoplasma affects wild and cultivated species of Prunus to different degrees, depending on their susceptibility. ‘Candidatus Phytoplasma prunorum’ is present in the four regions of Spain surveyed in this study (Aragon, Catalonia, Extremadura and Valencia) with a variable incidence. Results showed that ‘Ca. Phytoplasma prunorum’ was detected in all of the cultivated Prunus species studied, except P. avium and P. dulcis, and was widespread in Spain. The most affected species was P. salicina, with symptoms including early bud break and blooming, leaf curling and yellowing, collapse, and a major decrease in production. In some plots in the Baix Llobregat area of Barcelona province (Catalonia), the incidence of ESFY on P. salicina was as high as 80%. The insect vector, Cacopsylla pruni, was present in all four of the regions studied, with the highest captures in yellow sticky traps in Catalonia on P. mahaleb and in Extremadura in peach orchards. In Baix Llobregat, large populations of C. pruni were present on infected P. mahaleb bushes, and with high infection rates. This was a key factor in the local pathogenic cycle that caused a major ESFY outbreak in the nearby P. salicina orchards. In the Ebro valley (Lleida and Aragon) and Valencia, the surveys showed very low incidences of the disease and low C. pruni populations.     

Current situation and characterization of Pseudomonas syringae pv. actinidiae on kiwifruit in Galicia (northwest Spain)

Bacterial canker of kiwifruit, caused by Pseudomonas syringae pv. actinidiae (Psa), is a disease that is spreading rapidly in several kiwifruit‐producing countries, causing significant economic losses. In 2011, it was detected for the first time in Spain, in the south of Galicia (northwest Spain). Kiwifruit orchards were therefore inspected and sampled in 2011 and 2012 to determine the pathogen distribution, and the isolates obtained were characterized by morphology, fatty acids profile, biochemical tests and molecular techniques. Isolates were obtained from Actinidia deliciosa ‘Hayward’ (from leaves, canes, flower buds, fruits and roots), from A. deliciosa ‘Summer’, from Actinidia chinensis ‘Jin Tao’ (from canes and leaves) and from A. chinensis pollinator ‘Belén’ (from canes). Results of the analysis of the cfl gene (phytotoxin production‐related), the tox–argK gene cluster and phylogenetic analysis of the cts gene demonstrated that all Psa isolates from northwest Spain correspond to the Psa3 population, which includes strains of haplotype 2. This is the first record of Psa3 and haplotype 2 in Spain.     

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.     

Bleeding canker of horse chestnut (Aesculus hippocastanum) in Ireland: incidence,severity and characterization using DNA sequences and real‐time PCR

A survey of bleeding canker disease, caused by Pseudomonas syringae pv. aesculi, was undertaken across Ireland. Incidence has become severe and can be considered epidemic, as 61% of the 1587 horse chestnut trees surveyed showed symptoms of the disease. Bacteria were isolated from a sample of trees and characterized using gyrBDNA sequencing. DNA was also extracted directly from wound tissue. The Irish P. syringae pv. aesculi genotype was identical to genotypes previously sequenced with gyrB from the UK and some other locations in Europe. Real‐time PCR, using existing primers and a newly designed, more pathovar‐specific primer set, was assessed for use in disease screening. With molecular screening, a total of 11 trees from a sample of 55 tested positive for P. syringae pv. aesculi in Ireland. It was more efficient to extract DNA directly from wound tissue, especially fresh bark, for disease detection than to undertake bacterial isolation with subsequent molecular analysis. A further set of sequencing primers was developed for the amplification of the gyrB gene from P. syringae pv. aesculi and their specificity was shown using a diverse sample of bacterial isolate DNAs. The study also isolated and identified other bacterial species from diseased material; some of these are known pathogens (Brenneria nigrifluens, P. marginalis and P. syringae) or have previously been identified as potentially beneficial endophytes of host trees (Erwinia billingiae, E. tolentana, P. fluorescens, P. putida and Raoultella).     

A simple detached leaf assay provides rapid and inexpensive determination of pathogenicity of Pythium isolates to ‘all year round’ (AYR) chrysanthemum roots

A detached leaf assay was developed to determine the pathogenicity of Pythium isolates to cut‐flower chrysanthemum roots. Leaves from young plants were excised and inoculated by insertion of a plug of mycelium into a slit cut in the excised petiole. After incubation leaves were assessed for presence and extent of necrosis. Necrosis indicated pathogenicity and was consistently confirmed by comparisons with whole plant inoculations. The rate of necrosis spread also gave some indication of virulence. Isolates of Pythium sylvaticum, P. ultimum and HS group were the most virulent, with a mean rate of spread of 14·6 mm per day, significantly (P < 0·05) faster than the mean rate of spread, 1·6 mm per day, of less virulent isolates. Less virulent isolates included P. irregulare, P. oligandrum and P. aphanidermatum. The latter was unexpected, as P. aphanidermatum is an important species in pythium root rot epidemics in chrysanthemums elsewhere. The value of the detached leaf assay for screening large numbers of isolates was demonstrated in a survey of isolates from clinic samples from chrysanthemum nurseries and in a series of dilution‐plating experiments looking at numbers of Pythium propagules in commercial chrysanthemum beds showing root rot. In the survey, the predominant pathogenic species was identified as P. sylvaticum and the most likely source of infection was contaminated soil as opposed to blocking media or irrigation water, whilst in soil colonization studies the use of detached leaf assays demonstrated a relationship between pathogenic inoculum concentration in soil and the expression of root rot symptoms.