Identification of Monilia species in Tibet and characterization of M. yunnanensis in China

Samples of peach and plum fruits with brown rot symptoms were collected from Tibet in 2019 and 2020, and the causal agent was identified as Monilia yunnanensis, which represents the first characterization of Monilia spp. on peach and plum in Tibet. Morphological investigation showed that some conidia from naturally diseased fruits were larger than those observed in previously isolated M. yunnanensis. Some conidia of M. yunnanensis isolates from Tibet produced more than two, even up to six germ tubes from different parts of each conidium, instead of one or two germ tubes developing from the pointy sides of each conidium. The alignment of ribosomal internal transcribed spacer region (ITS) sequences revealed that some isolates from Tibet displayed a mutation at the 374th position from adenine (A) to cytosine (C). Although above-mentioned differences were observed between isolates from Tibet and other regions, phylogenetic analysis indicated that all of the M. yunnanensis isolates from different stone fruits and different regions in China were clustered together without obvious genetic differentiation. These results revealed that hosts and geographical environments did not play a major role in the evolution of M. yunnanensis.

A Duplex-Droplet Digital PCR Assay for Simultaneous Quantitative Detection of Monilinia fructicola and Monilinia laxa on Stone Fruits

Brown rot, caused by different Monilinia species, is a most economically important disease of pome and stone fruits worldwide. In Europe and in Italy, the quarantine pathogen M. fructicola was recently introduced and rapidly spread and, by competing with the main indigenous species Monilinia fructigena and Monilinia laxa, caused relevant changes in Monilinia populations. As a result, in most areas, the pathogen almost replaced M. fructigena and now coexists with M. laxa. The availability of specific and easy-of-use quantification methods is essential to study the population dynamics, and in this work, a new method for the simultaneous quantification of M. fructicola and M. laxa based on droplet digital PCR (ddPCR) technique was established. Under the optimized reaction conditions, consisting of 250/500 nM of primers/probe sets concentration, 58°C as annealing temperature and 50 PCR cycles, the duplex-ddPCR assay was 200-fold more sensitive than duplex-real-time quantitative PCR (qPCR) assay, quantifying < 1 copy μL–1 of target DNA in the PCR mixture. The results obtained with the validation assay performed on apricot and peach fruits, artificially inoculated with conidial suspensions containing different ratios of M. fructicola and M. laxa, showed a high correlation (R2 = 0.98) between the relative quantity of DNA of the two species quantified by ddPCR and qPCR and a more accurate quantification by ddPCR compared to qPCR at higher concentrations of M. fructicola. The herein described method represents a useful tool for the early detection of Monilinia spp. on stone fruits and for the improving knowledge on the epidemiology of brow rot and interactions between the two prevalent Monilinia species.

Varietal Wealth of Prunus Species

Genus Prunus includes all the stone fruits (peach, nectarine, plum, apricot, almond and cherry) comprise around 98 species and classified under three subgenera namely: Amygdalus (peaches, nectraine and almonds), Prunophora (plums and apricots) and Cerasus (cherries). Genus Prunus have attained a prime position among all the temperate fruit crops as delicious edible drupe, and many species have ornamental values as well. Major species of importance are Prunus persica (peach), Prunus armeniaca (apricot), Prunus salicina (Japanese plum), Prunus domestica (European plum), Prunus americana (American plum), Prunus avium (Sweet cherry), Prunus cerasus (Sour cherry), Prunus dulcis (almond), Prunus ceracifera (Cherry plum), Prunus mira (Behmi), Prunus cerasoides (Wild Himalayan cherry), Prunus mahaleb (Mahaleb cherry) etc. Interspecific hybrids namely: plumcots, pluots and apriums also produce very delicious edible fruits. Commercial cultivars of different stone fruits are J H Hale, Cresthaven, Flordasun, Florda Prince, Elberta, Glohaven, July Elberta, Redhaven, Kanto 5, Sun Haven etc. of peaches, Fantasia, Mayfire, Red Gold, Snow Queen etc. belongs to nectarine, Turkey, Charmagz, Perfection, St. Ambroise, Royal, New Castle etc. are apricots, Santa Rosa, Black Beauty, Kelsey, Green Gage, Methley, Satsuma, Frontier, Burbank etc. are plums, Regina, Burlat, Lapins, Kordia, Stella, Bing, Van, Black Heart, Compact Lambert, Compact Stella etc. are cherries, and California Paper Shell, IXL, Mission, Nonpareil, Drake, Ne Plus Ultra, Pranyaj, Merced etc. are almonds.

First Report of Shot-hole on Flowering Cherry Caused by Burkholderia contaminans and Pseudomonas syringae pv. syringae

The shot-hole disease (SH) is one of the most common and important diseases affecting the flowering cherry (FC; Prunus × yedoensis Matsumura; ‘Somei-yoshino’) trees in South Korea every year, resulting in premature defoliation and reduced flowering in the following year. However, pathogens associated with the disease remain unknown, which has rendered disease management challenging. Here, the pathogens associated with SH, their biochemical characteristics, and their host range were elucidated. Detached leaf and in planta assays revealed that two biofilm-forming bacteria, namely Burkholderia contaminans (Bc) and Pseudomonas syringae pv. syringae (Pss), caused SH of FC trees. These pathogens were recorded for the first time as the causes of SH of FC trees in South Korea. Additionally, the two pathogens induced similar disease symptoms in several stone fruits belonging to the genus Prunus, including peach (P. persica), plum (P. salicina), and apricot (P. mume), with peach being the most susceptible. These results indicate that Bc and Pss caused SH on FC trees and presented a broad spectrum of hosts. Furthermore, Xanthomonas arboricola pv. pruni, the causative agent of leaf spot on stone fruits, incited brown spots and shot holes on FC leaves. Therefore, FC trees are susceptible to infections by various pathogenic bacteria, including Bc, Pss, and Xap. These findings will be of great importance as a reference for effective management of SH in the face of possible cross-infection between Prunus species in the future.

Comparative Genomics Used to Predict Virulence Factors and Metabolic Genes among Monilinia Species

Brown rot, caused by Monilinia spp., is among the most important diseases in stone fruits, and some pome fruits (mainly apples). This disease is responsible for significant yield losses, particularly in stone fruits, when weather conditions favorable for disease development appear. To achieve future sustainable strategies to control brown rot on fruit, one potential approach will be to characterize genomic variation among Monilinia spp. to define, among others, the capacity to infect fruit in this genus. In the present work, we performed genomic and phylogenomic comparisons of five Monilinia species and inferred differences in numbers of secreted proteins, including CAZy proteins and other proteins important for virulence. Duplications specific to Monilinia were sparse and, overall, more genes have been lost than gained. Among Monilinia spp., low variability in the CAZome was observed. Interestingly, we identified several secondary metabolism clusters based on similarity to known clusters, and among them was a cluster with homology to pyriculol that could be responsible for the synthesis of chloromonilicin. Furthermore, we compared sequences of all strains available from NCBI of these species to assess their MAT loci and heterokaryon compatibility systems. Our comparative analyses provide the basis for future studies into understanding how these genomic differences underlie common or differential abilities to interact with the host plant.

Whole Genome Sequence of Wilsonomyces Carpophilus, the Causal Agent of Shot Hole of Stone Fruits: Insights Into Secreted Proteins of a Necrotrophic Fungal Repository

Wilsonomyces carpophilus is a necrotrophic plant pathogenic fungus with a wide host range infecting all stone fruits such as peach, plum, apricot and cherry, and almonds among the nut crops. Necrotrophs are more devastating with a complex pathogenicity mechanism and least known effector repositories. Here, we report a 29.9 megabase draft genome assembly of W. carpophilus. We explored the hybrid technology of Illumina HiSeq and PacBio sequencing technologies to get the unbiased results of sequence reads. We aligned short Illumina reads against the long PacBio reads. A total of 10,901 protein-coding genes were predicted that includes varied set of genes such as HET genes, cytochrome-p450 genes, kinases etc. We mined 2851 simple sequence repeats (SSRs) in the genome assembly. We also predicted the diverse inventory of secretory proteins, transporters, primary and secondary metabolic enzymes. A total of 225 secreted proteins, hydrolases, polysaccharide-degrading enzymes, esterolytic, lipolytic and proteolytic enzymes were the most significant proteins reflecting the necrotrophic lifestyle of the W. carpophilus. We also identified 146 tRNAs and 52 rRNAs in the pathogen genome.