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.

Field-based recombinase polymerase amplification and lab-based qPCR assays for detection of Helicoverpa armigera.

Helicoverpa armigera (Hübner) is a major crop pest native to Europe, Asia, Australia, and Africa which has recently invaded South America and has caused billions of dollars in agricultural losses. Because of challenges in differentiating between H. armigera and Helicoverpa zea (Boddie), a closely related species native to North and South America, genetic tests have previously been developed to detect H. armigera DNA in pooled samples of moth legs. In this study, a field-based recombinase polymerase amplification (RPA) assay using a lateral flow strip and a qPCR melt curve assay were developed for specific detection of H. armigera DNA in pooled moth samples. In addition, a crude DNA extraction protocol for whole moths was developed to allow rapid preparation of DNA samples. The RPA field test was able to detect ≥ 10 pg of purified H. armigera DNA and the crude DNA of one H. armigera sample in a background of 999 H. zea equivalents. The qPCR assay was able to detect ≥ 100 fg of purified H. armigera DNA and the crude DNA of one H. armigera sample in a background of up to 99,999 H. zea equivalents. Both RPA and qPCR assays detected H. armigera in the crude DNA extracted in the field from a pool of one H. armigera moth and 999 H. zea moths. These newly developed molecular assays to detect H. armigera will contribute to large-scale surveillance programs of H. armigera.

Parsimonious root systems and better root distribution can improve biomass production and yield of soybean.

Enhancing the acquisition of belowground resources has been identified as an opportunity for improving soybean productivity worldwide. Root system architecture is gaining interest as a selection criterion in breeding programs for enhancing soil resource acquisition and developing climate-resilient varieties. Here we are presenting two novel characteristics of soybean root system architecture that improve aboveground growth and yield. Eleven selected soybean genotypes were tested under rain-fed conditions in 2019 and 2020 at two locations in South Carolina, in which one of the locations was characterized by compacted soils. The elite SC breeding line SC07-1518RR, exotic pedigree line N09-12854, and slow wilting line N09-13890 were superior genotypes in terms of biomass production, seed yield, and/or water use efficiency. Genotypes N09-12854 and N09-13890 demonstrated reduced root development (based on total root count and length), likely to restrict belowground growth and allocate more resources for shoot growth. This characteristic, which can be referred as a parsimonious root phenotype, might be advantageous for soybean improvement in high-input production systems (characterized by adequate fertilizer application and soil fertility) that exist in many parts of the world. Genotype SC07-1518RR exhibited a similar strategy: while it maintained its root system at an intermediate size through reduced levels of total root count and length, it selectively distributed more roots at deeper depths (53-70 cm). The increased root distribution of SC07-1518RR at deeper depths in compacted soil indicates its root penetrability and suitability for clayey soils with high penetration resistance. The beneficial root phenotypes identified in this study (parsimonious root development and selective root distribution in deeper depths) and the genotypes that possessed those phenotypes (SC07-1518RR, N09-12854, and N09-13890) will be useful for breeding programs in developing varieties for optimal, drought, and compacted-soil conditions.