Development of a Long-Amplicon Propidium Monoazide-Quantitative PCR Assay for Detection of Viable Xanthomonas arboricola pv. pruni Cells in Peach Trees.

Bacterial spot is one of the most serious diseases of peach caused by the pathogen Xanthomonas arboricola pv. pruni (XAP), leading to early defoliation and unmarketable fruit. The pathogen can overwinter in peach twigs and form spring cankers, which are considered the primary inoculum source for early season leaf and fruitlet infection. The amount of overwintering bacterial inoculum plays a critical role for the bacterial spot development, but no reliable quantification method is available. Thus, we developed a long-amplicon propidium monoazide (PMA)-quantitative PCR (qPCR) assay for specific detection of viable XAP cells. The optimized PMA-qPCR assay used 20 µM of PMAxx for pure bacterial suspensions and 100 µM for peach twig tissues. The Qiagen Plant Pro Kit with an additional lysozyme digestion step was the DNA extraction protocol that yielded the best detection sensitivity with the bacteria-spiked peach twig extracts. The PMA-qPCR assay was tested with different mixtures of viable and heat-killed XAP cells in pure bacterial suspensions and bacteria-spiked peach twig tissues. The results showed that this assay enabled sensitive, specific, and accurate quantification of viable XAP cells as low as 103 CFU/ml with the presence of up to 107 CFU/ml of dead XAP cells, while suppressing the amplification of DNA from dead cells. For mixtures of viable and dead cells, the PMA-qPCR results were linearly correlated with the predicted concentrations of viable XAP (R2 > 0.98). Thus, the PMA-qPCR assay will be a suitable tool for quantifying overwintering XAP population on peach trees.

First Report of Xanthomonas campestris Causing Leaf Blight on Buttercup (Ranunculus asiaticus) in South Carolina, USA.

Buttercup (Ranunculus asiaticus L.) is a popular and high value ornamental species grown in landscapes and gardens and as cut flowers. It is mostly cultivated in Europe, the Mediterranean, and the Americas (Beruto and Debergh, 2004). In January 2022, leaf blight was observed on approximately 24 of forty 4-month-old R. asiaticus plants grown in a high tunnel at a cut flower farm located in Anderson County, South Carolina, USA. Symptoms included irregular, vein-limited, and necrotic leaf lesions and yellowing. Some lesions had a chlorotic halo. Two diseased plants were submitted to the Clemson University Plant and Pest Diagnostic Clinic. Symptomatic leaves were surface sterilized with 10% bleach for 1 min and rinsed in sterile water. Small leaf portions (1 × 1 cm2) were excised from the margin of lesions. They were macerated in 500 µl of sterile water and incubated at room temperature for 10 min. A loopful of suspension was streaked on nutrient agar (NA). Slightly convex, yellowish-mucoid colonies appeared after incubation at 28°C for 48 h. Two isolates, 23A and 23B, from two plants were obtained by transferring single colonies to new NA plates. Both isolates were identified as X. campestris (probability values > 0.8) using a Biolog Microbial Identification System (GEN III Microplate; Identification Database v.2.8.0.15G). PCR amplification of these two isolates were performed for housekeeping genes gyrB, rpoD, and dnaK (Young et al. 2008). The amplicon sequences (GenBank accession nos.: OR101193 and OR101194 [dnaK]; OR101195 and OR101196 [gyrB]; OR101197 and OR101198 [rpoD]) were identical between the two isolates based on sequence alignment in MEGA11 (Tamura et al. 2021). Nucleotide BLAST of these three genes showed 94.6 to 98.9% identity (dnaK: 912 of 922 bp; gyrB: 827 of 839 bp; rpoD: 803 of 849 bp) with 100% coverage with the Xanthomonas campestris pv. campestris type strain (AE008922). A neighbor joining phylogenetic tree with the concatenated sequences of these three genes showed that 23A and 23B had the closest match with X. campestris pv. campestris. However, these two isolates tested negative in the probe-based qPCR assay specific for X. campestris pv. campestris with only the positive control amplified (Köhl et al. 2011), suggesting that they may belong to a new pathovar of X. campestris. To confirm the pathogenicity of these isolates, three healthy R. asiaticus plants each were spray inoculated with suspensions of 23A and 23B in sterile tap water until runoff (OD600 = 0.1, approx. 108 CFU/ml). The non-inoculated control plants received a sterile tap water spray. The experiment was conducted twice. All plants were maintained in a growth chamber at 24°C with 10-h photoperiod. Seven to 15 days after inoculation, necrotic lesions with chlorotic halo and leaf yellowing, similar to those observed in the field, were observed on inoculated plants, while the non-inoculated control plants remained symptomless. Koch's postulates were fulfilled by reisolating the bacteria from the symptomatic plants and confirming the bacterial identity with the sequence analysis described above. The disease was first reported in California in 1996 (Azad et al. 1996) but to the best of our knowledge has not been reported again in the United States. This is the first report of X. campestris causing bacterial leaf blight in R. asiaticus in South Carolina. Since more than 50% of the flower producers/farmers grow Ranunculus in South Carolina, further work is necessary to determine how widespread the disease is and its economic impact.

Identification and Chemical and Biological Management of Phytopythium vexans, the Causal Agent of Phytopythium Root and Crown Rot of Woody Ornamentals.

Soilborne diseases caused by pathogens such as Phytophthora, Rhizoctonia, Fusarium, Verticillium, and Pythium species are the most important diseases of woody ornamentals. Ginkgo (Ginkgo biloba) and red maple (Acer rubrum 'October Glory') plants grown in containers and fields in Tennessee showed root and crown rot symptoms with dark brown to black lesions in 2017 and 2018. The objective of this research was to isolate and identify pathogens affecting ginkgo and red maple plants in Tennessee nurseries and to develop fungicide/biofungicide management recommendations for nursery producers. Isolations were made from the infected roots. Several Phytophthora-like colonies with spherical zoospores, filamentous to globose oogoni, and whitish mycelium were isolated on V8-PARPH medium. To confirm identity, total genomic DNA was extracted, followed by sequence analysis of the internal transcribed spacer regions, large subunit of nuclear rRNA, and cytochrome c oxidase subunits I and II of mitochondrial DNA. Based on morphological and molecular analysis, Phytopythium vexans was described as a causal agent of crown and root rot from the infected ginkgo and red maple plants. To complete Koch's postulates, a pathogenicity test was performed by drenching 100 ml of V8 agar medium slurry of Phytopythium vexans inoculum on 1-year-old potted ginkgo plant root systems as well as red maple October Glory. Necrotic lesion development was observed in the root system 45 days after inoculation and Phytopythium vexans was reisolated from the roots of both ginkgo and red maple. All control ginkgo and red maple plants remained disease free and no pathogen was reisolated. In addition, the efficacy of fungicides, biofungicides, fertilizer, and host plant defense inducers (traditionally recommended for management of oomycete diseases) for control of Phytopythium crown and root rot was evaluated on ginkgo and red maple October Glory seedlings in greenhouse and field trials. Fungicides such as Empress Intrinsic, Pageant Intrinsic, Segovis, and Subdue MAXX were effective in both greenhouse and field trials, and the biofungicide Stargus reduced disease severity caused by pathogen Phytopythium vexans on ginkgo and red maple plants in greenhouse trials. These results will help nursery producers make proper management decisions for newly reported Phytopythium crown and root rot disease of ginkgo and red maple plants.

First Report of Phytopythium vexans Causing Root and Crown Rot on Flowering Cherry in Tennessee.

Flowering cherry (Prunus serrulata Lindl. 'Kwanzan') rooted cuttings grown in propagation beds containing 40% coarse sand and 60% ground pine bark in a commercial propagation nursery in Warren County, Tennessee were exhibiting root and crown rot in December 2016. Dark brown to black soft lesions were observed in the roots as well as the crown region of flowering cherry rooted cuttings and those rooted cuttings were non-marketable due to lesions. Disease incidence was approximately 60% of 10,000 plants. Phytophthora ImmunoStrip test (Agdia Inc., Elkhart, IN, USA) was performed and the test result was positive. Diseased plant tissues were surface sterilized with 70% ethanol and washed twice with distilled water. Culturing the affected root and crown parts (1 cm pieces) on V8-PARPH, an oomycete-selective medium consistently yielded whitish radiate mycelial growth pattern with spherical zoospores, filamentous to globose oogoni, elongated, and cylindrical antheridia with constrictions (De Cock et al., 2015) after 7 days of incubation at 25°C in a 12-h fluorescent light and dark cycle, which is the typical morphology of Phytopythium vexans (de Bary) Abad, de Cock, Bala, Robideau, Lodhi & Lévesque. To confirm pathogen identity, total DNA was extracted using the UltraClean Microbial DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA) directly from a 3-day old culture of isolate (FBG2017010) on V8 medium. The internal transcribed spacer (ITS) and 28S large subunit of ribosomal RNA, and cytochrome c oxidase subunit I (CoxI) of mitochondrial DNA (mtDNA) genes/ region were amplified by PCR using the primer pairs ITS1/ ITS4 (White et al., 1990), NL1/ NL4 (Baten et al., 2014), and Levup and Fm85mod (Robideau et al., 2011), respectively. The PCR products were sequenced and the sequences (GenBank accession nos. MT533275, MT533451, and MT547980) were compared to the voucher specimens. They were 99.23, 99.60, and 98.92% similar to those of P. vexans isolates in the NCBI database (HQ643400, KR092144, and HQ708996, respectively). To complete Koch's postulates, 'Kwanzan' flowering cherry rooted cuttings grown on propagation substrate (10 cm pot containing 1 kg sterilized 40% coarse sand and 60% ground pine bark) were inoculated with identified pathogen and observations were taken on root rot disease symptoms. Five plants were inoculated with 100 ml of pathogen agar-slurry (1 plate of a 7-day old culture of isolate FBG2017010/1 L of sterilized water), and five control plants were drenched with agar slurry. The plants were maintained in the greenhouse condition (day/night temperature of 26/24°C), and irrigated twice a day for 2 min by overhead irrigation system. After 2 weeks, dark brown to black necrotic root lesions developed on all inoculated cuttings and P. vexans was consistently re-isolated from the inoculated plants. The morphology of the pathogen isolated on the V8-PARPH medium was identical to the original isolate. All control plants remained symptom-free and P. vexans was not isolated from the root tissue. To our knowledge, this is the first report of P. vexans causing root and crown rot in 'Kwanzan' flowering cherry in Tennessee, which can be a potential threat for the nursery crop production. The identification of P. vexans, the causal agent of Phytopythium root and crown rot is important in determination and implementation of effective management strategies.