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.

Complete Genome Sequence Resource for Xanthomonas fragariae Causing Crown Infection Pockets in Strawberry

Xanthomonas fragariae (X. fragariae ) is a global quarantine pathogen, which typically inflicting angular leaf spots. In the present study, we report a new 4.11 Mb high-quality genome sequence of X. fragariae YL19. YL19 can make the strawberry plants have the typical angular leaf spot symptom and have crown infection pockets symptom in China. This new symptom has not been reported in other X. fragariae. Compared with typical X. fragariae strains, including PD885, NBC2815, PD5205, Fap21, and Fap29, the genome and plasmid in YL19 were smaller in size, which lacked 109 coding genes and has more CAZymes genes and secondary metabolism genes. The YL19 genome ought to clarify the molecular mechanisms of genome evolution, host adaptation, and pathological process of X. fragariae and the improvement of strawberry management strategies.

Complete genome and plasmid sequence data of three Xanthomonas arboricola pv. corylina strains, the bacterium responsible for bacterial blight of hazelnut

Xanthomonas arboricola pv. corylina is the causal agent of bacterial blight of hazelnut. The bacterium is listed as A2 quarantine pathogen in Europe since 1978 and on the Regulated Non-Quarantine Pest (RNQP) list since 2020. Three strains from various geographic regions and isolated at different times were sequenced using a hybrid approach with short- and long-read technologies to generate closed genome and plasmid sequences in order to better understand the biology of this pathogen.

Microbe Profile: Xylella fastidiosa – a devastating agricultural pathogen with an endophytic lifestyle

Xylella fastidiosa is a vector-borne plant vascular pathogen that has caused devastating disease outbreaks in diverse agricultural crops worldwide. A major global quarantine pathogen, X. fastidiosa can infect hundreds of plant species and can be transmitted by many different xylem sap-feeding insects. Several decades of research have revealed a complex lifestyle dependent on adaptation to the xylem and insect environments and interactions with host plant tissues.

Pear blister canker viroid.

Pear blister canker disease, first reported in France in the 1960's, is caused by pear blister canker viroid (PBCVd). Diseases can occur in several cultivars of pear (Pyrus communis), its major host, but most pear cultivars are tolerant and do not produce symptoms of infection. PBCVd was initially reported in pear and quince, followed by wild pear and nashi and can experimentally infect species in other genera (Chaenomeles, Cydonia, Sorbus, Malus). Transmission through mechanical inoculation from pruning tools and grafting to infective propagative materials is a potential pathway for spread; no animal vector is known and it is not known to be seed transmitted. PBCVd has been reported in several European countries, Malta, Tunisia, Turkey, Australia, Japan, China, and the Americas; the geographic distribution may be underestimated because of symptomless infections in certain hosts. PBCVd was placed on the A1 list in Canada in 1995, listed as a quarantine pest in the United States in 1989, is regarded as quarantine pathogen for Australia, and is listed in a certification scheme to produce clean Pyrus and Cydonia sp. planting material in an OEPP/EPPO Bulletin in 1999 (OEPP/EPPO, 1999).

Pear blister canker viroid.

Pear blister canker disease, first reported in France in the 1960's, is caused by pear blister canker viroid (PBCVd). Diseases can occur in several cultivars of pear (Pyrus communis), its major host, but most pear cultivars are tolerant and do not produce symptoms of infection. PBCVd was initially reported in pear and quince, followed by wild pear and nashi and can experimentally infect species in other genera (Chaenomeles, Cydonia, Sorbus, Malus). Transmission through mechanical inoculation from pruning tools and grafting to infective propagative materials is a potential pathway for spread; no animal vector is known and it is not known to be seed transmitted. PBCVd has been reported in several European countries, Malta, Tunisia, Turkey, Australia, Japan, China, and the Americas; the geographic distribution may be underestimated because of symptomless infections in certain hosts. PBCVd was placed on the A1 list in Canada in 1995, listed as a quarantine pest in the United States in 1989, is regarded as quarantine pathogen for Australia, and is listed in a certification scheme to produce clean Pyrus and Cydonia sp. planting material in an OEPP/EPPO Bulletin in 1999 (OEPP/EPPO, 1999).

Cherry leaf roll virus (walnut ringspot).

Seed transmission of CLRV is a threat to gene bank contamination. CLRV-contaminated vegetative propagation material and seeds are prone to extremely long-distance transport. Due to this risk potential, CLRV is included in the list of plant viruses that should be closely monitored during sanitary production of propagative material, especially for walnut and olive trees (Bassi and Martelli, 2003). CLRV is treated as an A2 quarantine pathogen in Rubus in the EPPO region, a virus-free certification scheme for Rubus was developed by OEPP/EPPO (1994). In cherry (Kegler et al., 1972; Bush, 2005), walnut (Mircetich et al., 1980; Delbos et al., 1983; Nemeth et al., 1990) and olive production areas, CLRV infections are consistently occurring (Langer et al., 2010; Büttner et al., 2011); crop losses due to CLRV infections were reported for cherry (Kegler et al., 1972; Bush, 2005), walnut (Mircetich et al., 1980; Delbos et al., 1983; Nemeth et al., 1990) and raspberry (Jones and Wood, 1978).In birch species native to Fennoscandia virus-like symptoms on leaves (vein banding, leaf roll, mottling), partially adherent with progressive loss of vitality or death of twigs and branches have been spreading rapidly since 2002 with up to 85% of the tested trees being infected with CLRV (Jalkanen et al., 2007; von Bargen et al., 2009a).

Apiognomonia erythrostoma (cherry leaf scorch).

A. erythrostoma is a perithecial ascomycete known primarily from Europe, although it has also been reported from eastern Asia. The early spotting of leaves and fruits of Prunus species, particularly cherry and apricot [Prunus armeniaca], can result in significant defoliation and loss of yield in certain years when weather conditions are favourable for infection by airborne ascospores. Although there is no record of introduction of the fungus to new areas, which would most likely require transport of trees still bearing infected leaves and fruit, some countries do list it as a quarantine pathogen.

The Role of NbTMP1, a Surface Protein of Sporoplasm, in Nosema Bombycis Infection

Background: Nosema bombycis is a unicellular eukaryotic pathogen of the silkworm, Bombyx mori, and a hazard to the silkworm industry. Because of its long incubation period and horizontal and vertical transmission, it is a quarantine pathogen in sericulture. The microsporidian life cycle includes a dormant extracellular phase and intracellular proliferation phase. The proliferation period is the most active period of the microsporidian. This period lacks spore wall protection and may be the most susceptible stage for control. Results: The N. bombycis protein (NBO_76g0014) was identified as a transmembrane protein and named NbTMP1. It is not homologous with proteins from other microsporidia and species. NbTMP1 has a transmembrane region of 23 amino acids at the N-terminus. Indirect immunofluorescence analysis (IFA) results suggest that NbTMP1 is secreted on the plasma membrane as the spores develop. Western blot and qRT-PCR analysis showed that NbTMP1 expressed in all development stages of N. bombycis in infected cells and in the silkworm midgut. Down-regulation of NbTMP1 expression resulted in significant inhibition of N. bombycis proliferation. Conclusions: We confirmed that NbTMP1 is a membrane protein of N. bombycis. Reduction of the transcription level of NbTMP1 significantly inhibited N. bombycis proliferation, and this protein may be a target for the selective breeding of N. bombycis resistant silkworm strains.

Widespread occurrence of 'Candidatus Phytoplasma ulmi' in elm species in Germany

Background: Candidatus Phytoplasma ulmi' is the agent associated with elm yellows and has been categorised in the European Union as a quarantine pathogen. For central and northern European countries, information on the occurrence and distribution of the pathogen and its impact on elms is scarce, so a survey of native elm trees has been conducted in Germany. Results: About 6,500 samples from Ulmus minor , Ulmus laevis and Ulmus glabra , were collected nationwide. Phytoplasma detection was performed by applying a universal 16Sr DNA-based quantitative PCR (qPCR) assay and a novel ' Ca. P. ulmi' specific qPCR assay targeting the 16S-23S spacer region. Both assays revealed that 28% of the samples were infected by ‘ Ca. P. ulmi’, but infection rates of the elm species and regional incidences differed. The phytoplasma presence in the trees was not correlated to disease-specific symptoms. The survey identified a regional disparity of infection which was high in east, south and central Germany, whereas only a few infected sites were found in the western and northern parts of the country. Monitoring the seasonal titre of ‘Ca. P. ulmi’ in an infected tree by qPCR revealed a high colonisation in all parts of the tree throughout the year. Conclusions: ‘ Ca. P. ulmi’ is widely present in elms in Germany. The rare occurrence of symptoms indicates either a high degree of tolerance in elm populations or a low virulence of pathogen strains enabling high infection rates in a long-living host.