N. W. Schaad

Real-time PCR and its application for rapid plant disease diagnostics

Rapid-cycle real-time polymerase chain reaction (PCR) methods may revolutionize the manner in which plant pathogens are identified and diseases are diagnosed. As the genomics age progresses and more and more DNA sequence data become available, highly specific primers and fluorescent probe sequences can be designed to yield target amplicons to unique regions of a pathogens genome. Portable real-time PCR instruments described here are now allowing for diagnostic assays to be run directly in the field or at remote locations other than the standard diagnostic laboratory. Rapid real-time PCR diagnosis can result in appropriate control measures and (or) eradication procedures more quickly and accurately than traditional methods of pathogen isolation. Disease losses are minimized and control costs reduced. Advantages and disadvantages of rapid real-time PCR for the detection of bacterial, fungal, and viral plant pathogens are described.

Xylella fastidiosa subspecies: X. fastidiosa subsp piercei, subsp. nov., X. fastidiosa subsp. multiplex subsp. nov., and X. fastidiosa subsp. pauca subsp. nov.

Xylella fastidiosa, a fastidious bacterium causing disease in over 100 plant species, is classified as a single species, although genetic studies support multiple taxons. To determine the taxonomic relatedness among strains of X. fastidiosa, we conducted DNA-DNA relatedness assays and sequenced the 16S-23S intergenic spacer (ITS) region using 26 strains from 10 hosts. Under stringent conditions (Tm -15 degrees C), the DNA relatedness for most X. fastidiosa strains was *70%. However, at high stringency (Tm -8 degrees C), three distinct genotypes (A, B, and C) were revealed. Taxon A included strains from cultivated grape, alfalfa, almond (two), and maple, interrelated by 85% (mean); taxon B included strains from peach, elm, plum, pigeon grape, sycamore, and almond (one), interrelated by 84%; and taxon C included only strains from citrus, interrelated by 87%. The mean reciprocal relatedness between taxons A and B, A and C, and B and C, were 58, 41, and 45%, respectively. ITS results also indicated the same grouping; taxons A and B, A and C, and B and C had identities of 98.7, 97.9, and 99.2%, respectively. Previous and present phenotypic data supports the molecular data. Taxon A strains grow faster on Pierces disease agar medium whereas B and C strains grow more slowly. Taxon B and C strains are susceptible to penicillin and resistant to carbenicillin whereas A strains are opposite. Each taxon can be differentiated serologically as well as by structural proteins. We propose taxons A, B, and C be named X. fastidiosa subsp. fastidiosa [correction] subsp. nov, subsp. multiplex, subsp. nov., and subsp. pauca, subsp. nov., respectively. The type strains of the subspecies are subsp. fastidiosa [correction] ICPB 50025 (= ATTC 35879T and ICMP 15197), subsp. multiplex ICPB 50039 (= ATTC 35871 and ICMP 15199), and subsp. pauca ICPB 50031 (= ICMP 15198).

Cultivation of 'Candidatus Liberibacter asiaticus', 'Ca. L. africanus', and 'Ca. L. americanus' associated with huanglongbing.

A new medium designated Liber A has been designed and used to successfully cultivate all three Candidatus Liberibacter spp., the suspect causative agents of huanglongbing (HLB) in citrus. The medium containing citrus vein extract and a growth factor sustained growth of Ca. Liberibacter spp. for four or five single-colony transfers before viability declined. Colonies, positive for Ca. L. asiaticus by a 16s-based rDNA real-time polymerase chain reaction (RT-PCR) assay and sequencing, were irregular-shaped, convex, and 0.1 to 0.3 mm after 3 to 4 days. Suspect Ca. L. asiaticus and Ca. L. americanus cells were observed in infected tissue and on agar culture by scanning electron microscopy. The cells were ovoid to rod shaped, 0.3 to 0.4 by 0.5 to 2.0 microm, often with fimbriae-like appendages. Two strains of Ca. L. asiaticus and one of Ca. L. americanus grown on Liber A medium were pathogenic on citrus and could be isolated from noninoculated tissues of inoculated trees and seedlings 9 and 2 months later, respectively. The identity was confirmed by RT-PCR and 16s rDNA sequencing. This is the first report of the cultivation and pathogenicity of Ca. L. asiaticus and Ca. L. americanus associated with symptoms of HLB.

Evaluation of Proposed Amended Names of Several Pseudomonads and Xanthomonads and Recommendations

ABSTRACT In 1980, over 90% of all plant-pathogenic pseudomonads and xanthomonads were lumped into Pseudomonas syringae and Xanthomonas campestris, respectively, as pathovars. The term pathovar was created to preserve the name of plant pathogens, but has no official standing in nomenclature. Proposals to elevate and rename several pathovars of the genera Pseudomonas and Xanthomonas to the rank of species has caused great confusion in the literature. We believe the following changes have merit and expect to adopt them for publication in a future American Phytopathological Society Laboratory Guide for Identification of Plant Pathogenic Bacteria. Upon review of published data and the Rules of The International Code of Nomenclature of Bacteria, we make the following recommendations. We reject the proposal to change the name of P. syringae pvs. phaseolicola and glycinea to P. savastanoi pvs. phaseolicola and glycinea, respectively, because both pathogens are easily differentiated phenotypically from pv. savastanoi and convincing genetic data to support such a change are lacking. We accept the elevation of P. syringae pv. savastanoi to the rank of species. We accept the reinstatement of X. oryzae to the rank of species with the inclusion of X. oryzicola as a pathovar of X. oryzae and we accept the species X. populi. We agree with the elevation of the pvs. cassavae, cucurbitae, hyacinthi, pisi, and translucens to the rank of species but not pvs. melonis, theicola, and vesicatoria type B. We recommend that all type A X. vesicatoria be retained as X. campestris pv. vesicatoria and all type B X. vesicatoria be named X. exitiosa. We reject the newly proposed epithets arboricola, bromi, codiaei (poinsettiicola type B), hortorum, sacchari, and vasicola and the transfer of many pathovars of X. campestris to X. axonopodis. The proposed pathovars of X. axonopodis should be retained as pathovars of X. campestris.

A real-time BIO-PCR assay for detection of Ralstonia solanacearum race 3, biovar 2, in asymptomatic potato tubers

Ralstonia solanacearum, the causal agent of brown rot of potato, can often be carried latently in seed potato tubers. We designed real-time PCR (polymerase chain reaction) primers and probe and a highly sensitive BIO-PCR assay for specific detection of the strains of race 3, biovar (bv.) 2 of R. solanacearum in asymptomatic potato tubers. The biovar-specific primers and probe reacted with all 17 strains of bv. 2 of R. solanacearum tested, including 12 from potato and 5 from geranium. None of the other 35 strains of R. solanacearum reacted, including 4 strains of bv. 1 from potato or the closely related blood disease bacterium from banana. None of 13 other bacteria reacted, including 5 Erwinia and 2 Clavibacter species from potato. Using undiluted potato tuber extract, inoculated with R. solanacearum bv. 2, as few as 30 cells/mL of extract could be detected. Two of 14 naturally infected potato tubers with no disease symptoms were positive by the newly described real-time BIO-PCR assay, whereas none were positive with a standard real-time PCR assay. This is the first report of the detection of R. solanacearum bv. 2 in asymptomatic, latently infected potato tubers by PCR. The real-time assay is very simple and much less time consuming than classical PCR.

Emergence of Acidovorax avenae subsp. citrulli as a Crop Threatening Disease of Watermelon and Melon

Acidovorax avenae subsp. citrulli (Pseudomonas pseudoalcaligenes subsp. citrulli), the causal agent of a watermelon seedling blight and fruit blotch (WFB), has emerged as a serious seedborne pathogen of watermelon and melons world-wide. The disease was first reported in 1965 in seedlings of several USDA Plant Introduction (PI) lines in Georgia. The pathogen was described 13 years later in diseased seedlings from two of the same PI seed sources in which Web and Goth had originally identified the disease. The disease remained known only in seedlings at the introduction station until Wall and Santos isolated a non-fluorescent pseudomonad from rotting fruit in Guam in 1987 and concluded it was the same organism. WFB was reported in Florida in 1989 and a year later emerged as a major economic disease of watermelon in the Southeast and Midwest USA. Since then, WFB has been reported in Turkey, Brazil, Australia, Japan, Korea, Thailand, and China infecting cantaloupe, melon, honeydew, pumpkin, and citron under field conditions. Because the original seedling strains were reported as giving a negative tobacco hypersensitive reaction and failed to rot fruit like the field disease strains, some have suggested that the originally described organism was not the same organism as that causing WFB. DNA fingerprinting of a collection of 121 strains using pulse field gel electrophoresis revealed considerable genetic diversity. All strains typed into two groups; over 80% were in Group I and the remaining, including the original type strain, in Group II. Control of the disease is based primarily on use of pathogen-free seed. Although attempts have been made to develop a routine seed assay to detect the organism in seeds, no reliable method is available. Contaminated seeds remain a problem for the seed industry.

Genetic Diversity in Populations of Xanthomonas campestris pv. campestris in Cruciferous Weeds in Central Coastal California

ABSTRACT Xanthomonas campestris pv. campestris (X. campestris) infects a large number of cruciferous plants, including weeds. California has one of the largest and most diverse populations of wild cruciferous plants in the world. Although considerable information is available on the genetic diversity of X. campestris in commercial crop plants, nothing is known about the diversity in strains infecting weeds. To assess the genetic diversity among strains of X. campestris in weeds in noncultivated and cultivated areas, strains of the pathogen were isolated from populations of cruciferous weeds growing in coastal valley crop-production sites and from remote nonproduction sites along the California central coast. Results of fingerprinting over 68 strains using amplified fragment length polymorphism along with representative strains by sequence analysis showed the presence of seven genotypes. Genotypes A and B were limited to coastal sites; genotypes C, D, and E were from inland cultivated sites; and genotypes F and G were present in both coastal noncultivated and inland cultivated sites. Crop strains were grouped outside any weed strain group and were separated from the weed strains and other pathovars of X. campestris. These results revealed, for the first time, that strains of X. campestris present in noncultivated coastal weed populations generally were unique to a site and genetically distinct from strains present in populations of weeds in crop-production areas located nearby.

Use of PCR for rapid identification of Acidovorax avenae and A. avenae subsp. citrulli.

The species Acidovorax avenae contains bacteria pathogenic to several crops: A. a. subsp. avenae infects oats, corn, rice, millet, and sugarcane; subsp. citrulli infects watermelon and melon; and subsp. cattleyae infects orchid. Because these bacteria are closely related their proper identification requires expensive, time-consuming pathogenicity tests. To develop PCR assays for rapid identification of all species and subsp. citrulli, we sequenced the intergenic spacer region of the 16S–23S rDNA and designed avenae-specific and subsp. citrulli-specific classical primers and real-time primers and probes. Classical avenae-specific primers Oafl/Oarl amplified a DNA product from all 46 strains of subsp. avenae originating from foxtail, oats, corn, rice, sugarcane, and millet; the type strains of subsp. cattleyae from orchid; and 11 strains of subsp. citrulli from watermelon. Real-time avenae-specific primers Oafl/Oar2 and probe AaP1 reacted with all 40 strains of subsp. avenae, the type strain of subsp. cattleyae, and 8 strains of subsp. citrulli. Classical citrulli-specific primers Aacf2/Aacr2 and real-time primers Aacf3/Aacr2 and probe AaP2 amplified a PCR product from all 11 strains of subsp. citrulli, but not from any other strain of subsp. avenae. None of over 50 other bacteria tested generated a PCR product with either classical primers or real-time primers and probe. The species-specific classical primers Oafl/Oarl and real-time primers Oafl/Oar2 and probe AaP1 should prove useful for rapid identification of A. avenae strains from all hosts, whereas the citrulli-specific classical primers Aacf2/Aacr2 and real-time primers Aacf3/Aacr2 and probe AaP2 should be useful for specific identification of subsp. citrulli.

Genetic Characterization and Diversity of Rathayibacter toxicus

ABSTRACT Rathayibacter toxicus is a nematode-vectored gram-positive bacterium responsible for a gumming disease of grasses and production of a highly potent animal and human toxin that is often fatal to livestock and has a history of occurring in unexpected circumstances. DNA of 22 strains of R. toxicus from Australia were characterized using amplified fragment length polymorphism (AFLP) and pulsed-field gel electrophoresis (PFGE). AFLP analysis grouped the 22 strains into three genetic clusters that correspond to their geographic origin. The mean similarity between the three clusters was 85 to 86%. PFGE analysis generated three different banding patterns that enabled typing the strains into three genotypic groups corresponding to the same AFLP clusters. The similarity coefficient was 63 to 81% for XbaI and 79 to 84% for SpeI. AFLP and PFGE analyses exhibited an analogous level of discriminatory power and produced congruent results. PFGE analysis indicated that the R. toxicus genome was represented by a single linear chromosome, estimated to be 2.214 to 2.301 Mb. No plasmids were detected.

Detection of Ralstonia solanacearum in ginger rhizomes by real-time PCR

Bacterial wilt of ginger (Zingiber officinale), caused by Ralstonia solanacearum (Rs), has emerged as an important disease of ginger production in Thailand and throughout Asia. Real-time PCR assays were developed for detection of Rs in ginger rhizomes. A unique 329-bp DNA fragment from Rs biovar 4 from ginger was identified using amplified fragment length polymorphisms, and the nucleotide sequence was determined. PCR primer and probe sequences were designed for real-time PCR assays and screened against 86 strains of Rs. The primers RSAF1 and RSAR1 and probe RSP1 were shown to react with all strains of Rs race 1 biovars 3 and 4 but not biovars 1 and 2. Real-time TaqMan® PCR protocols were developed for two real-time PCR platforms, the ABI 7700 sequence detection system (Applied Biosystems, Foster City, Calif.) and the portable Smart Cycler (Cepheid, Sunnyvale, Calif.). A comparison between classical real-time PCR and real-time BIO-PCR protocols, using 15 asymptomatic ginger rhizomes collected from different fields and markets, showed that 13 and 9 were positive by standard PCR and BIO-PCR, respectively. This is the first description of a real-time PCR assay capable of detecting Rs in asymptomatic ginger rhizomes and the first report of Rs in asymptomatic ginger rhizomes being sold in markets in Thailand.