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.     

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.     

SNP-based genotyping and whole-genome sequencing reveal previously unknown genetic diversity in Xanthomonas vasicola pv. musacearum,causal agent of banana xanthomonas wilt,in its presumed Ethiopian origin

For decades, Xanthomonas vasicola pv. musacearum (Xvm) has been an economically important bacterial pathogen on enset in Ethiopia. Since 2001, Xvm has also been responsible for significant losses to banana crops in several East and Central African countries, with devastating consequences for smallholder farmers. Understanding the genetic diversity within Xvm populations is essential for the smart design of transnationally reasoned, durable, and effective management practices. Previous studies have revealed limited genetic diversity in Xvm, with East African isolates from banana each falling into one of two closely related clades previously designated as sublineages SL 1 and SL 2, the former of which had also been detected on banana and enset in Ethiopia. Given the presumed origin of Xvm in Ethiopia, we hypothesized that both clades might be found in that country, along with additional genotypes not seen in Central and East African bananas. Genotyping of 97 isolates and whole-genome sequencing of 15 isolates revealed not only the presence of SL 2 in Ethiopia, but additional diversity beyond SL 1 and SL 2 in four new clades. Moreover, SL 2 was detected in the Democratic Republic of Congo, where previously SL 1 was the only clade reported. These results demonstrate a greater range of genetic diversity among Xvm isolates than previously reported, especially in Ethiopia, and further support the hypothesis that the East/Central Africa xanthomonas wilt epidemic has been caused by a restricted set of genotypes drawn from a highly diverse pathogen pool in Ethiopia.     

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.     

PCR‐based identification of cacao black pod causal agents and identification of biological factors possibly contributing to Phytophthora megakarya's field dominance in West Africa

Among the Phytophthora species that cause black pod of cacao, P. megakarya is the most virulent, posing a serious threat to cacao production in Africa. Correct identification of the species causing the black pod and understanding the virulence factors involved are important for developing sustainable disease management strategies. A simple PCR‐based species identification method was developed using the species‐specific sequences in the ITS regions of the rRNA gene. A phylogenetic tree generated for 119 Phytophthora isolates, based on the 60S ribosomal protein L10 gene and rDNA sequence, verified the PCR‐based identification assay and showed high interspecific variation among the species causing black pod. Phytophthora megakarya isolates were uniformly virulent in an assay using susceptible cacao pod husks inoculated with zoospores, while the P. palmivora isolates showed greater divergence in virulence. The virulence of P. megakarya was associated with earlier production of sporangia and an accelerated induction of necrosis. While zoospore germ tubes of both species penetrated pods through stomata, only P. megakarya produced significant numbers of appressoria. A hypersensitive‐like response was observed when attached SCA‐6 pods were inoculated with P. palmivora. SCA‐6 pods became vulnerable to P. palmivora when wounded prior to zoospore inoculation. Phytophthora megakarya was more aggressive than P. palmivora on attached SCA‐6 pods, causing expanding necrotic lesions with or without wounding. Phytophthora megakarya is predominant in the Volta region of Ghana and it remains to be seen whether it can displace P. palmivora from cacao plantations of Ghana as it has in Nigeria and Cameroon.     

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.     

High throughput real‐time RT‐PCR assays for specific detection of cassava brown streak disease causal viruses,and their application to testing of planting material

Cassava brown streak disease (CBSD) caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) is causing severe losses in cassava production in Kenya, Tanzania and Uganda. Two real‐time RT‐PCR assays based on TaqMan chemistry capable of detecting and distinguishing these two viruses are described. These assays were used to screen 493 cassava samples collected from western and coastal Kenya, the main cassava regions of Uganda and inland Tanzania. Both viruses were found in all three countries and across regions therein. Association of CBSD leaf symptom status with CBSV and UCBSV assay results was weak, confirming the need for a diagnostic assay. For leaf samples that were observed with CBSD‐like leaf symptoms but shown as CBSV and UCBSV negative by the RT‐PCR assay, deep sequencing using a Roche 454 GS‐FLX was used to provide additional evidence for the absence of the viruses. The probability of the CBSD associated diagnostics detecting a single CBSV or UCBSV positive sample amongst other non‐CBSD samples was modelled. The results of this study are discussed in the context of the application of diagnostics of CBSD‐associated viruses under the Great Lakes Cassava Initiative and the need to minimize the risk of further spread of the viruses with cassava multiplication material. It is shown that high throughput testing undertaken at Fera of 300 cassava leaves taken from fields for seed multiplication, when analysed in pools of 10, has given a 95% probability of detecting 1% infected plants in the field.     

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

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

Panel of real‐time PCRs for the multiplexed detection of two tomato‐infecting begomoviruses and their cognate whitefly vector species

A new approach for the simultaneous identification of the viruses and vectors responsible for tomato yellow leaf curl disease (TYLCD) epidemics is presented. A panel of quantitative multiplexed real‐time PCR assays was developed for the sensitive and reliable detection of Tomato yellow leaf curl virus‐Israel (TYLCV‐IL), Tomato leaf curl virus (ToLCV), Bemisia tabaci Middle East Asia Minor 1 species (MEAM1, B biotype) and B. tabaci Mediterranean species (MED, Q biotype) from either plant or whitefly samples. For quality‐assurance purposes, two internal control assays were included in the assay panel for the co‐amplification of solanaceous plant DNA or B. tabaci DNA. All assays were shown to be specific and reproducible. The multiplexed assays were able to reliably detect as few as 10 plasmid copies of TYLCV‐IL, 100 plasmid copies of ToLCV, 500 fg B. tabaci MEAM1 and 300 fg B. tabaci MED DNA. Evaluated methods for routine testing of field‐collected whiteflies are presented, including protocols for processing B. tabaci captured on yellow sticky traps and for bulking of multiple B. tabaci individuals prior to DNA extraction. This work assembles all of the essential features of a validated and quality‐assured diagnostic method for the identification and discrimination of tomato‐infecting begomovirus and B. tabaci vector species in Australia. This flexible panel of assays will facilitate improved quarantine, biosecurity and disease‐management programmes both in Australia and worldwide.     

Development of a grower‐conducted inoculum detection assay for management of grape powdery mildew

Management of grape powdery mildew (Erysiphe necator) and other polycyclic diseases often relies on calendar‐based pesticide application schedules that assume the presence of inoculum. An inexpensive, loop‐mediated isothermal amplification (LAMP) assay was designed to quickly detect airborne inoculum of E. necator to determine when to initiate a fungicide application programme. Field efficacy was tested in 2010 and 2011 in several commercial and research vineyards in the Willamette Valley of Oregon from pre‐bud break to véraison. In each vineyard, three impaction spore traps were placed adjacent to the trunk. One trap was maintained and used by the grower to conduct the LAMP assay (G‐LAMP) on‐site and the other two traps were used for laboratory‐conducted LAMP (L‐LAMP) and quantitative PCR assay (qPCR). Using the qPCR as a gold standard, L‐LAMP was comparable with qPCR in both years, and G‐LAMP was comparable to qPCR in 2011. Latent class analysis indicated that qPCR had a true positive proportion of 98% in 2010 and 89% in 2011 and true negative proportion of 96% in 2010 and 64% in 2011. An average of 3·3 fewer fungicide applications were used when they were initiated based on spore detection relative to the grower standard practice. There were no significant differences in berry or leaf incidence between plots with fungicides initiated at detection or grower standard practice plots, suggesting that growers using LAMP to initiate fungicide applications can use fewer fungicide applications to manage powdery mildew compared to standard practices.     

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.     

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.     

Evaluation of organic amendments from agro‐industry waste for the control of verticillium wilt of olive

Biological control of plant diseases using soil amendments such as animal manure and composted materials can minimize organic waste and has been proposed as an effective strategy in crop protection. In this study, 35 organic amendments (OAs) and 16 compost mixtures were evaluated against Verticillium dahliae by assessing both the antagonistic effect on the mycelial growth of two representative isolates of V. dahliae and the effect on the reduction of microsclerotia viability of the pathogen in naturally infested soil. Eleven OAs and five compost mixtures showed a consistent inhibition effect in in vitro sensitivity tests, with solid olive‐oil waste compost one of the most effective. Therefore, a bioassay with olive plants was conducted to evaluate the suppressive effect against V. dahliae of these selected OAs and compost mixtures. Significant reduction in the severity of the symptoms of V. dahliae indicates the potential use of grape marc compost (100% disease severity reduction) and solid olive‐oil waste, combined with other OAs. Microorganism mixtures and dairy waste OAs had a potential suppressive effect when they were combined with compost, showing a 73% and 63% disease severity reduction, respectively. A mixture of agro‐industrial waste with other biological control agents is a promising strategy against verticillium wilt of olive. To the authors' knowledge, this is the first report on the effectiveness of compost extracts (compost teas) on the inhibition of natural microsclerotia of V. dahliae, and also on verticillium wilt suppression in olive with solid olive‐oil waste.     

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.     

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.     

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.     

Development of a real‐time PCR method for the detection of the dagger nematodes Xiphinema index,X. diversicaudatum,X. vuittenezi and X. italiae,and for the quantification of X. index numbers

The ectoparasitic dagger nematodes Xiphinema index and Xiphinema diversicaudatum, often at low numbers in the soil, are vectors of grapevine nepoviruses, which cause huge agronomical problems for the vineyard industry. This study reports a method, based on real‐time PCR, for the specific detection of these species and of the closely related non‐vector species Xiphinema vuittenezi and Xiphinema italiae. Specific primers and TaqMan probes were designed from the ribosomal DNA internal transcribed spacer 1 (ITS1), enabling the specific detection of single individuals of each of the X. index, X. diversicaudatum, X. italiae and X. vuittenezi species whatever the nematode population. The specificity of detection and absence of false positive reaction were confirmed in samples of each species mixed with the three other Xiphinema species or mixed with nematodes representative from other genera (non‐plant‐parasitic Dorylaimida, Longidorus sp., Meloidogyne spp., Globodera spp. and Pratylenchus sp.). The method was shown to be valid for the relative quantification of X. index numbers through its use, from crude nematode extracts of soil samples, in a greenhouse assay of grapevine accessions ranging from highly susceptible to resistant. As an alternative to time‐consuming microscopic identification and counting, this real‐time PCR method will provide a fast, sensitive and reliable diagnostic and relative quantification technique for X. index nematodes extracted from fields or controlled conditions.     

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.     

Development of a real‐time PCR assay for Pseudomonas cichorii,the causal agent of midrib rot in greenhouse‐grown lettuce,and its detection in irrigating water

Midrib rot is an emerging disease in greenhouse production of lettuce caused by Pseudomonas cichorii, and probably introduced through contaminated irrigation water. Concentrations of 100 CFU mL^?1 are enough to induce the typical midrib rot symptoms. A sensitive real‐time PCR assay was developed, based on a 90‐bp amplicon from the pathogenicity gene cluster hrcRST and a Taqman Minor Groove Binding probe. Specificity of the assay was tested with 39 P. cichorii strains, including the type strain, and 89 strains from 83 other Pseudomonas species. The relationship between detection signals and P. cichorii DNA concentrations was linear over 6‐logs. Detection threshold with excellent reproducibility was 500 fg of DNA or about 70 genome copies. Sample preparation and DNA isolation were optimized to allow detection in 1 L water samples. The assay was first evaluated with greenhouse irrigation water spiked with serial dilutions of P. cichorii. The calculated cell numbers obtained with real‐time PCR were 10‐fold lower than plate counts of actual spiked cells. However, the assay consistently detected 100 CFU per reaction, corresponding to the detection of 1 CFU mL^?1 of irrigation water, which is well below the concentration needed for midrib rot infection. Finally, the assay proved to be valuable for detecting infective P. cichorii concentrations in the irrigation water of a commercial lettuce production greenhouse.