Development of a specific molecular tool for detecting Xanthomonas campestris pv. musacearum

A specific and rapid diagnostic tool has been developed to detect Xanthomonas campestris pv. musacearum, the causal agent of bacterial wilt of banana. PCR primers were developed from intergenic regions of X. campestris pv. musacearum following its partial sequence. A total of 48 primers were tested for specificity to X. campestris pv. musacearum strains collected from various regions in Uganda. These were also tested for specificity against related Xanthomonas species from the vasicola group, Xanthomonas species pathogenic to other crops, and against those existing saprophytically on banana plants. Seven primer sets (Xcm12, Xcm35, Xcm36, Xcm38, Xcm44, Xcm47 and Xcm48) were found to be very specific to X. campestris pv. musacearum. These primer sets directed the amplification of the expected product for all 52 strains of X. campestris pv. musacearum collected from different locations in Uganda. No amplification products were obtained with unrelated phytopathogenic bacteria or endophytic/epiphytic bacteria from banana. A detection limit of 10³ CFU mL^?1 corresponding to about four cells per PCR reaction was observed when X. campestris pv. musacearum cells were used for all the seven primer sets. The DNA samples from symptomless plant tissues also tested positive with primer set Xcm38. The specific PCR method described here is a valuable diagnostic tool which can be used to detect the pathogen at early stages of infection and monitor disease.     

Multiplex PCR for specific and robust detection of Xanthomonas campestris pv. musacearum in pure culture and infected plant material

The present study developed a pathovar‐specific PCR for the detection of Xanthomonas campestris pv. musacearum (Xcm), the cause of banana xanthomonas wilt, by amplification of a 265‐bp region of the gene encoding the general secretion pathway protein D (GspD). A distinct DNA fragment of the expected size was amplified from genomic DNA from all of 12 Xcm isolates tested and no amplification of DNA was observed from other xanthomonads or plant‐associated bacteria, including the two closely related species Xanthomonas vasicola pv. holcicola and Xanthomonas axonopodis pv. vasculorum. The Xcm‐specific PCR was successfully multiplexed with internal control primers targeting 16S rDNA for application on DNA from bacterial cultures and with primers targeting plant mitochondrial 26S rDNA for application on DNA extracted from plant material. Diagnostic discrimination of healthy and infected plants was subsequently demonstrated in tests on artificially inoculated screenhouse cultivars of banana and field bananas with and without symptoms sampled from different parts of Uganda. This study therefore demonstrated a robust and specific Xcm diagnostic tool with the added advantage of applying internal PCR controls for direct quality assessment of results.     

Genomics‐informed design of loop‐mediated isothermal amplification for detection of phytopathogenic Xanthomonas arboricola pv. pruni at the intraspecific level

The objective of this study was to develop a rapid, sensitive detection assay for the quarantine pathogen Xanthomonas arboricola pv. pruni, causal agent of stone fruit bacterial spot, an economically important disease of Prunus spp. Unique targets were identified from X. arboricola pv. pruni genomes using a comparative genomics pipeline of other Xanthomonas species, subspecies and pathovars, and used to identify specific diagnostic markers. Loop‐mediated isothermal amplification (LAMP) was then applied to these markers to provide rapid, sensitive and specific detection. The method developed showed unrivalled specificity with the 79 tested strains and, in contrast to previously established techniques, distinguished between phylogenetically close subspecies such as X. arboricola pv. corylina. The sensitivity of this test is comparable to that of a previously reported TaqMan^? assay at 10³ CFU mL^?1, while the unrivalled speed of LAMP technology enables a positive result to be obtained in <15 min. The developed assay can be used with real‐time fluorescent detectors for quantitative results as well as with DNA‐staining dyes to function as a simplified strategy for on‐site pathogen detection.     

Sources of resistance in Musa to Xanthomonas campestris pv. musacearum,the causal agent of banana xanthomonas wilt

It is claimed that, with the exception of Musa balbisiana, all banana varieties are susceptible to bacterial wilt caused by Xanthomonas campestris pv. musacearum (Xcm). Despite being resistant to Xcm infection, M. balbisiana is not preferred for breeding because it belongs to the BB genome subgroup, while most edible bananas are of the A genome. To identify potential sources of resistance to Xcm, 72 banana accessions representing the Musa genetic diversity were evaluated in an outdoor confined potted trial. The midribs of the youngest leaf of 3-month-old banana plants were inoculated with 10⁸ CFU mL⁻¹ of Xcm isolate USY13P, and symptom development assessed weekly for 4 months. Results confirmed that M. balbisiana genotypes are indeed resistant to Xcm. Varieties within the Musa acuminata subsp. zebrina (AA) set were further identified as potentially useful sources of Xcm resistance. These findings reveal the potential to develop banana and plantain varieties with tolerance to Xcm.     

Systemicity of Xanthomonas campestris pv. musacearum and time to disease expression after inflorescence infection in East African highland and Pisang Awak bananas in Uganda

Banana xanthomonas wilt (XW) caused by Xanthomonas campestris pv. musacearum (Xcm) attacks all banana cultivars. Xcm in inflorescence‐infected Pisang Awak plants with wilting male bud bracts is restricted to the upper parts of the true stem; therefore, cutting these plants at the pseudostem base has been recommended to prevent further Xcm spread. In order to fine‐tune existing control strategies, this study examined the movement of Xcm into plants and mats, in relation to disease incubation period. Mature Pisang Awak and East African highland (AAA‐EA) plants were inoculated with Xcm through abscission wounds of female bracts, male bud bracts, male flowers, a combination of male bud bracts and flowers, and by cutting male buds with a contaminated machete. Thirty plants per genotype and treatment were monitored for 24 months for disease symptoms. An additional 68 AAA‐EA and 33 Pisang Awak plants were sampled weekly to assess the rate of Xcm spread within the plants. All floral entry points resulted in disease, with the highest incidence in combined male bract and male flower abscission wound inoculations. The study confirmed the systemicity of Xcm, with the pathogen able to live within the mat for long periods (5–16 months) without causing disease. Reliance on disease symptom expression to manage XW is therefore not sufficient. The long incubation period in lateral shoots may explain the current resurgence of the disease in locations where the disease was thought to have been successfully eradicated.     

Development of a semiselective medium for isolating Xanthomonas campestris pv. musacearum from insect vectors, infected plant material and soil

A semiselective medium was developed for isolating Xanthomonas campestris pv. musacearum ( Xcm ) from infected banana plants, soil and insect vectors. The new medium was named cellobiose-cephalexin agar (CCA) and it contained (L⁻¹): 1 g yeast extract, 1 g glucose, 1 g peptone, 1 g NH₄Cl, 1 g MgSO₄·7H₂O, 3 g K₂HPO₄, 1 g beef extract, 10 g cellobiose, 14 g agar, 40 mg cephalexin, 10 mg 5-fluorouracil and 120 mg cycloheximide. The medium was evaluated for selectivity using 21 bacterial isolates and for plating efficiency using Xcm . The bacterial isolates included a soilborne Xanthomonas species and three pathogenic Xanthomonas strains that infect cassava, cabbage and beans. Although the plating efficiency of Xcm on CCA was lower (59%) than on non-selective yeast extract peptone glucose agar (YPGA), its selectivity was significantly higher, averaging 60 and 82%, when isolating from banana fruits and soil, respectively. CCA was also superior when isolating Xcm from insect vectors, with selectivity of 48–75%, compared with 8–17% on YPGA. Xanthomonas campestris pv. phaseoli did not grow on CCA, while X. campestris pv. campestris and X. axonopodis pv. manihotis grew, but their colonies were smaller than those of Xcm . Twenty-nine out of 33 suspected Xcm strains isolated from plants, soil and insects using CCA were pathogenic when inoculated onto banana plants, indicating that CCA can be a reliable tool in isolating Xcm populations. The medium should prove useful in studies on ecology, epidemiology and management of the banana bacterial wilt pathogen that is currently ravaging bananas in East and Central Africa.     

Comparative and collaborative studies for the validation of a nested PCR for the detection of Xanthomonas axonopodis pv. dieffenbachiae from Anthurium samples

Efficient control of Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of anthurium bacterial blight, requires sensitive and reliable diagnostic tools. The European standard EN ISO 16140:2003 has been followed to compare a nested PCR assay (N‐PCR) to a reference method (isolation and serological identification of bacterial colonies) and to other alternative serological detection methods. The evaluation was performed in two steps: a comparative study and a collaborative study involving 15 European laboratories. Although inclusivity was maximal (100%) for all methods, a maximal exclusivity was obtained only with N‐PCR followed by an enzymatic restriction digestion of the amplicons. Exclusivity indices of 90·6, 88·7 and 47·2% were found for indirect ELISA, immunofluorescence and double antibody sandwich ELISA, respectively. An exclusivity of 92·5% was obtained with the reference method, further increased to 100% if pathogenicity tests were performed as a supplemental assay. The best level of sensitivity (relative detection level) was obtained with the reference method followed by the N‐PCR assay. The N‐PCR performance in terms of relative accuracy, accordance and concordance was very similar to that of the reference method. Moreover, N‐PCR had undeniable advantages compared to the reference method (less labour‐intensive and less time‐consuming). In addition, post‐test probabilities of infection were calculated to select the most appropriate detection scheme related to the prevalence of the pathogen. The N‐PCR assay has since been included in a revised version of the EPPO detection protocol.     

A quantitative real‐time PCR assay for detection of Colletotrichum lindemuthianum in navy bean seeds

Bean anthracnose is a seedborne disease of common bean (Phaseolus vulgaris) caused by the fungal pathogen Colletotrichum lindemuthianum. Using seed that did not test positive for the pathogen has been proven to be an effective strategy for bean anthracnose control. To quantify the extent of anthracnose seed infection, a real‐time PCR‐based diagnostic assay was developed for detecting C. lindemuthianum in seeds of the commercial bean class navy bean. The ribosomal DNA (rDNA) region consisting of part of the18S rDNA, 5^.8S rDNA, internal transcribed spacers (ITS) 1, 2 and part of the 28S rDNA of seven races of C. lindemuthianum, 21 isolates of Colletotrichum species and nine other bean pathogens were sequenced with the universal primer set ITS5/ITS4. Based on the aligned sequence matrix, one primer set and a probe were designed for a SYBR Green dye assay and a TaqMan MGB (minor groove binder) assay. The primer set was demonstrated to be specific for C. lindemuthianum and showed a high sensitivity for the target pathogen. The detection limit of both assays was 5 fg of C. lindemuthianum genomic DNA. To explore the correlation between the lesion area and the DNA amount of C. lindemuthianum in bean seed, seeds of the navy bean cultivar Navigator with lesions of different sizes, as well as symptomless seeds, were used in both real‐time PCR assays.     

Incomplete systemic movement of Xanthomonas campestris pv. musacearum and the occurrence of latent infections in xanthomonas wilt‐infected banana mats

Management of banana xanthomonas wilt (XW) (caused by Xanthomonas campestris pv. musacearum, Xcm) has been impeded by poor adoption of control options that are complex, cumbersome and costly. To improve XW management, this study investigated Xcm survival and latent infections in subsequent generations, survival of latently infected planting materials (suckers), incidence of latent infections in symptomless plants in mats having diseased plants, and XW status across farms and markets in districts previously devastated but currently endemic. On‐station experiments were protected from new infections. Latent bacteria at low levels were detected in up to 20% of the third generation suckers, with a significant (P < 0·05) reduction (43–20%) in subsequent generations. Only 3–6% of latently infected suckers succumbed to XW. Incidence of Xcm in symptomless suckers from farmers' fields (with up to 70% incidence) was low (3%) while it increased (8–25%) with disease severity in mats in controlled experiments. In the surveyed districts, incidence had significantly declined with yields observed to have recovered relative to earlier reports, although latent infections remained high. This study provides evidence that if new infections are prevented, fields with high XW incidence can be rejuvenated. It showed incomplete systemic movement of Xcm in mats coupled to a gradual decline of bacterial load in subsequent generations to levels that cannot initiate disease. These studies explain the current successes in farms practising single diseased plant removal instead of whole mat rouging, and gives hope to farmers lacking access to clean planting material.     

Development of a semi-selective medium for isolation of <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv<Emphasis Type="Italic">. musacearum</Emphasis> from banana plants

Banana Xanthomonas wilt, caused by Xanthomonas campestris pv. musacearum, is a new threat to banana cultivation in eastern Africa. The causal bacterium grows slowly in culture and is easily overgrown by contaminants. A selective culture medium for isolation of X. c. pv. musacearum will facilitate disease study. A medium that suppressed saprophytic growth and possessed diagnostic characters for the pathogen was developed. Various carbon sources were tested with two isolates of X. c. pv. musacearum, and sucrose was selected as main carbon source. The susceptibility of X. c. pv. musacearum and other bacterial strains was tested with 29 different antibiotics. Cephalexin and cycloheximide had no effect on X. c. pv. musacearum but cephalexin inhibited most of the saprophytes and cycloheximide inhibited the fungal contaminants. Based on these studies, we have developed a semi-selective medium YTSA-CC containing yeast extract (1%), tryptone (1%), sucrose (1%), agar (1.5%), cephalexin (50 mg l⁻¹) and cycloheximide (150 mg l⁻¹), pH 7.0. The pathogen X. c. pv. musacearum was easily identified as yellowish, mucoid and circular colonies on YTSA-CC medium. This simple semi-selective medium was effective for isolation of X. c. pv. musacearum from infected banana tissues and soil, and it should be a valuable tool in ecological and epidemiological studies.     

Silencing of Erwinia amylovora sy69 AHL‐quorum sensing by a Bacillus simplex AHL‐inducible aiiA gene encoding a zinc‐dependent N‐acyl‐homoserine lactonase

Quorum sensing in Gram‐negative bacteria is regulated by diffusible signal molecules called N‐acyl‐l ‐homoserine lactones (AHLs). These molecules are degraded by lactonases. In this study, six Bacillus simplex isolates were characterized and identified as a new quorum‐quenching species of Bacillus. An aiiA gene encoding an AHL‐lactonase was identified based on evidence that: (i) it showed high homology with other aiiA genes of Bacillus sp.; (ii) the deduced amino acid sequence contained two conserved regions, ¹⁰⁴SHLHFDH¹¹¹ and ¹⁶⁵TPGHTPGH¹⁷³, characteristic of the metallo‐β‐lactamase superfamily; and (iii) the protein had zinc‐dependent AHL‐degrading activity. Additionally, the expression of the aiiA gene was significantly up‐regulated by 3‐oxo‐AHL. The AHL‐lactonase inhibited multiplication of the 3‐oxo‐C6‐AHL‐producing plant pathogen Erwinia amylovora sy69 both in vitro and in planta. The results provide support for the use of the quorum‐quenching functionality of B. simplex in the integrated control of the devastating fire blight pathogen.     

Redefining the global populations of Pseudomonas syringae pv. actinidiae based on pathogenic,molecular and phenotypic characteristics

Knowing the population structure of a pathogen is fundamental for developing reliable phytosanitary legislation, detection techniques, and control strategies based on the actual aggressiveness and distribution of the pathogen. Currently, four populations of Pseudomonas syringae pv. actinidiae (Psa) have been described: Psa 1, Psa 2, Psa 3 and Psa 4. However, diagnostic assays specific for Psa populations do not detect Psa 4, the less virulent (LV) strains isolated in New Zealand. Similarly, multilocus sequence typing (MLST) of housekeeping genes, or broad Psa strain genome comparisons, revealed that Psa 4‐LV strains clustered separately from other Psa populations. In order to examine whether the placement of Psa 4 in the pathovar actinidiae was appropriate, various tests were carried out. It was shown that the Psa 4‐LV strains induced leaf and shoot wilting in Prunus cerasus, extensive necrotic lesions in Capsicum annuum fruits, and no significant symptoms in Actinidia deliciosa. Moreover, repetitive‐sequence PCR fingerprinting, type III secretion system effector protein genes detection and colony morphology clearly indicated the distinctiveness of Psa 4‐LV strains from the other three Psa populations. Rep‐PCR molecular typing revealed a high similarity of the Psa 4‐LV strains with members of Pseudomonas avellanae species. The Psa 4‐LV strains, most probably, belong to a new, still unnamed pathovar. It was concluded that the Psa 4‐LV strains isolated in New Zealand do not belong to the pathovar actinidiae, and, consequently, three Psa populations pathogenic to Actinidia spp. should currently include Psa 1, Psa 2 and Psa 3.     

Inference of the phylogenetic diversity and population structure of Xanthomonas campestris affecting Brassicaceae using a multilocus sequence typing‐based approach

Xanthomonas campestris pathovars are widely distributed throughout the globe and have a broad host range, causing severe economic losses in the food and ornamental crucifers markets. Using an approach based on multilocus sequence typing, phylogenetic diversity and population structure of a set of 75 Portuguese and other Xanthomonas campestris isolates from several cruciferous hosts were assessed. Although this population displayed a major clonal structure, neighbour‐net phylogenetic analysis highlighted the presence of recombinational events that may have driven the ecological specialization of X. campestris with different host ranges within the Brassicaceae family. A high level of genetic diversity within and among X. campestris pathovars was also revealed, through the establishment of 46 sequence types (STs). This approach provided a snapshot of the global X. campestris population structure in cruciferous host plants, correlating the existing pathovars with three distinct genetic lineages. Phylogenetic relationships between the founder genotype and remaining isolates that constitute the X. campestris pv. campestris population were further clarified using goeBURST algorithm. Identification of an intermediate link between X. campestris pv. campestris and X. campestris pv. raphani provided new insights into the mechanisms driving the differentiation of both pathovars. Wide geographic distribution of allelic variants suggests that evolution of X. campestris as a seedborne pathogen was not shaped by natural barriers. However, as Portuguese isolates encompass 26 unique STs and this country is an important centre of domestication of Brassica oleracea crops, a strong case is made for its role as a diversification reservoir, most probably through host–pathogen coevolution.     

Real‐time and qualitative PCR for detecting Pseudomonas syringae pv. actinidiae isolates causing recent outbreaks of kiwifruit bacterial canker

Since 2008, Pseudomonas syringae pv. actinidiae virulent strains (Psa‐V) have quickly spread across the main areas of kiwifruit (Actinidia deliciosa and A. chinensis) cultivation causing sudden and re‐emerging outbreaks of bacterial canker to both species. The disease caused by Psa‐V strains is considered worldwide as pandemic. Recently, P. syringae strains (ex Psa‐LV, now called PsD) phylogenetically related to Psa‐V have been isolated from kiwifruit, but cause only minor damage (i.e. leaf spot) to the host. The different biological significance of these bacterial populations affecting kiwifruit highlights the importance of having a diagnostic method able to detect Psa‐V, which is currently solely responsible for the severe damage to the kiwifruit industry. In order to improve the specific molecular detection of Psa‐V, a real‐time PCR assay has been developed based on EvaGreen chemistry, together with a novel qualitative PCR (PCR‐C). Both methods are based on specific primer sets for the hrpW gene of Psa. The real‐time PCR and PCR‐C were highly specific, detecting down to 50 and 200 fg, respectively, and were applied to a range of organs/tissues of kiwifruit with and without symptoms. These methods are important tools for both sanitary and certification programmes, and will help to avoid the spread of Psa‐V and to check possible inoculum sources. In addition to being used as routine tests, they will also enable the study of the biology of Psa‐V and the disease that it causes, whilst avoiding the detection of other populations of related P. syringae present in kiwifruit.     

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 Phytophthora conserved transposon‐like DNA element as a potential target for soyabean root rot disease diagnosis

A transposon‐like element, A3aPro, with multiple copies in the Phytophthora sojae genome, was identified as a suitable detection target for this devastating soyabean root rot pathogen. The PCR primers TrapF1/TrapR1 were designed based on unique sequences derived from the transposon‐like sequence. A 267‐bp DNA fragment was amplified using this primer pair, the specificity of which was evaluated against 118 isolates of P. sojae, 72 isolates of 25 other Phytophthora spp., isolates of Pythium spp. and isolates of true fungi. In tests with P. sojae genomic DNA, detection sensitivities of 10 pg and 10 fg DNA were achieved in standard PCR (TrapF1/TrapR1) and nested PCR (TrapF1/TrapR1 and TrapF2/TrapR2), respectively. Meanwhile, PCR with TrapF1/TrapR1 primers detected the pathogen at the level of a single oospore, and even one zoospore. These primers also proved to be efficient in detecting pathogens from diseased soyabean tissues, residues and soils. In addition, real‐time quantitative PCR (qPCR) assays coupled with the TrapF1/TrapR1 primers were developed to detect and quantify the pathogen. The results demonstrated that the TrapF1/TrapR1 and TrapF2/TrapR2 primer‐based PCR assay provides a rapid and sensitive tool for the detection of P. sojae in plants and in production fields.     

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.     

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.     

Efficacy of UV‐C radiation to reduce seedborne anthracnose (Colletotrichum acutatum) from Andean lupin (Lupinus mutabilis)

The potential of UV‐C radiation of Andean lupin (Lupinus mutabilis) seeds to eradicate seedborne infections of anthracnose caused by Colletotrichum acutatum was investigated. UV‐C doses from 0 to 691.2 kJ m^?2 (resulting from 0 to 96 h of exposure time) on disease incidence reduction and germination on artificially and naturally infected seed were evaluated. The degree of incidence reduction and seed germination was dependent on the dose of UV‐C. The UV‐C doses of 86.4 kJ m^?2 and higher reduced incidence from 6% to 7% to undetectable levels, but these UV‐C doses also reduced seed germination. UV‐C can deleteriously affect physiological processes and overall growth. To assess its impact, L. mutabilis seeds irradiated with UV‐C doses of 57.6 and 86.4 kJ m^?2 were grown. Seedlings grown from noninfected seed and UV‐C treated seed showed an increased concentration of chlorophyll and protein contents, as well as an increase in the activation of defence enzymes peroxidase and catalase, in comparison with plants grown from infected seed. UV‐C doses resulted in seed emergence and seedling dry weight rates that were similar to the noninfected control or better than the fungicide control. Moreover, 57.6 kJ m^?2 reduced transmission of the pathogen from seed to the plantlets by 80%, while 86.4 kJ m^?2 apparently eradicated the pathogen, under greenhouse conditions. The use of UV‐C, first reported here, is advantageous for controlling anthracnose in lupin.     

Highly specific assays to detect isolates of Pseudomonas syringae pv. actinidiae biovar 3 and Pseudomonas syringae pv. actinidifoliorum directly from plant material

Pseudomonas syringae pv. actinidiae (Psa) is responsible for bacterial canker of kiwifruit. Biovar 3 of Psa (Psa3) has been causing widespread damage to yellow‐ and green‐fleshed kiwifruit (Actinidia spp.) cultivars in all the major kiwifruit‐producing countries in the world. In some areas, including New Zealand, P. syringae pv. actinidifoliorum (Pfm), another bacterial pathogen of kiwifruit, was initially classified as a low virulence biovar of Psa. Ability to rapidly distinguish between these pathovars is vital to the management of bacterial canker. Whole genome sequencing (WGS) data were used to develop PCR assays to specifically detect Psa3 and Pfm from field‐collected material without the need to culture bacteria. Genomic data from 36 strains of Psa, Pfm or related isolates enabled identification of areas of genomic variation suitable for primer design. The developed assays were tested on 147 non‐target bacterial species including strains likely to be found in kiwifruit orchards. A number of assays did not proceed because although they were able to discriminate between the different Psa biovars and Pfm, they also produced amplicons from other unrelated bacteria. This could have resulted in false positives from environmental samples, and demonstrates the care that is required when applying assays devised for pure cultures to field‐collected samples. The strategy described here for developing assays for distinguishing strains of closely related pathogens could be applied to other diseases with characteristics similar to Psa.