Phytophthora cinnamomi is an ecologically and agriculturally significant plant pathogen. Early and accurate detection of P. cinnamomi is paramount to disease prevention and management. In this study, a loop-mediated isothermal amplification (LAMP) assay utilizing a new target gene Pcinn100006 identified from genomic sequence data was developed and evaluated for the detection of P. cinnamomi. This Pcinn100006 LAMP assay was found highly specific to P. cinnamomi. All 10 tested isolates of P. cinnamomi yielded positive results, whereas 50 isolates belonging to 16 other Phytophthora species, Globisporangium ultimum, and 14 fungal species lacked detection. This assay was 10 times more sensitive (100 pg in a 25-µl reaction mixture) than a conventional PCR assay (2 ng in a 50-µl reaction mixture) for detecting the genomic DNA of P. cinnamomi. In addition, it detected P. cinnamomi from artificially inoculated leaves of Cedrus deodara. Moreover, detection rates of P. cinnamomi using environmental DNAs extracted from 13 naturally infested rhizosphere samples were 100% in the Pcinn100006 LAMP assay versus 46% in the conventional PCR assay. Considering its higher accuracy and shorter time span, this Pcinn100006 LAMP assay is a promising diagnostic tool to replace conventional PCR-based and culture-dependent assays for screening of P. cinnamomi in regions at risk of infection or contamination.

A previously unknown Phytophthora species was isolated from irrigation water in Virginia, USA. This novel species produces abundant noncaducous and nonpapillate sporangia in soil water extract solution. It sometimes produces chlamydospores and hyphal swellings in aged cultures and in Petri’s solution. This species has optimum vegetative growth at 30 C and grows well at 35 C. The lowest and highest temperatures for growth are 5 and 40 C. All isolates examined in this study are compatibility type A1 and produce mostly plerotic oospores when paired with an A2 mating-type tester of P. cinnamomi. Sequence analyses of the rDNA internal transcribed spacer (ITS) regions and the mitochondrially encoded cytochrome c oxidase 1 (cox 1) gene placed this species in clade 9 of the genus Phytophthora. These characteristics support the description of this taxon as a new species for which we propose the name P. hydrogena sp. nov. Further phylogenetic and physiological investigations of clade 9 species revealed a high-temperature tolerant cluster including P. hydrogena, P. aquimorbida, P. hydropathica, P. irrigata, P. chrysanthemi, P. insolita, P. polonica and P. parsiana. These species all grow well at 35 C. The monophyly of the species in this heat-tolerant cluster except P. insolita and P. polonica is highly supported by the maximum-likelihood analyses of the ITS and cox 1 sequences.

Pitch canker caused by the fungus Fusarium circinatum is a damaging disease that affects pines in Europe, South Africa, and North America in both the southeast and west coast of the United States. Several countries, including China, have listed F. circinatum as a quarantine pathogen. Timely detection, an important pillar of the quarantine effort, can efficiently prevent the introduction of F. circinatum into new areas or facilitate management and eradication strategies in already infested sites. In this study, we developed an F. circinatum detection technique based on a combination of recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology (termed RPA-CRISPR/Cas12a). After obtaining DNA, this novel method can be utilized for the molecular identification of F. circinatum using the naked eye and can specifically detect F. circinatum at DNA concentrations as low as 200 fg within 30 min at 37°C. The system is sensitive for both standard laboratory samples and samples from the field. In summary, we have developed a simple, rapid, sensitive, unaided-eye visualization, RPA-CRISPR/Cas12a-based detection system for the molecular identification of F. circinatum that does not require technical expertise or expensive ancillary equipment.

Phytophthora cinnamomi is a devastating pathogen causing root and crown rot and dieback diseases of nearly 5000 plant species. Accurate and rapid detection of P. cinnamomi plays a fundamental role within the current disease prevention and management programs. In this study, a novel effector gene PHYCI_587572 was found as unique to P. cinnamomi based on a comparative genomic analysis of 12 Phytophthora species. Its avirulence homolog protein 87 (Avh87) is characterized by the Arg-Xaa-Leu-Arg (RxLR) motif. Avh87 suppressed the pro-apoptotic protein BAX- and elicitin protein INF1-mediated cell death of Nicotiana benthamiana. Furthermore, a recombinase polymerase amplification-lateral flow dipstick detection assay targeting this P. cinnamomi-specific biomarker was developed. While successfully detected 19 P. cinnamomi isolates of a global distribution, this assay lacked detection of 37 other oomycete and fungal species, including P. parvispora, a sister taxon of P. cinnamomi. In addition, it detected P. cinnamomi from artificially inoculated leaves of Cedrus deodara. Moreover, the RPA-LFD assay was found to be more sensitive than a conventional PCR assay, by detecting as low as 2 pg of genomic DNA in a 50-µL reaction. It detected P. cinnamomi in 13 infested soil samples, while the detection rate was 46.2% using PCR. Results in this study indicated that PHYCI_587572 is a unique biomarker for detecting P. cinnamomi. Although PHYCI_587572 was identified as an effector gene based on the RxLR motif of Avh87 and the avirulence activity on Nicotiana, its exact genetic background and biological function on the natural hosts of P. cinnamomi warrant further investigations.

Pitch canker caused by the fungus Fusarium circinatum is an important disease affecting pine trees in Europe and South Africa. Several countries, including China, have listed F. circinatum as a quarantine pathogen. Therefore, timely detection of F. circinatum could efficiently prevent its introduction into new areas or facilitate spread management in already infected sites. In this study, a recombinase polymerase amplification–lateral flow dipstick (RPA-LFD) assay was developed for rapid detection of F. circinatum based on a new target gene, Fcir2067, identified from whole-genome sequences. The assay was highly specific to F. circinatum. In fact, it exclusively detected F. circinatum isolates; 53 isolates of fungal and oomycete species and 2 nematodes of Bursaphelenchus xylophilus and B. mucronatus were not detected. By detecting as little as 10 pg of F. circinatum genomic DNA in a 50-µl reaction, the RPA-LFD assay was 10 times more sensitive than conventional PCR assays. F. circinatum was also detected in artificially inoculated pine needles of Cedrus deodara. These results demonstrated that the developed RPA-LFD assay has the potential for rapid detection of F. circinatum in regions at high risk of infection. The RPA-LFD assay might serve as an alternative method for the early detection of F. circinatum.

During a 2019–2020 survey of plant pathogenic oomycetes in Nanjing, China, a cluster of five adjacent Rhododendron pulchrum plants in Xuanwuhu Park exhibited symptoms including crown and root rot a...