Soil, rhizosphere, and root microbiome in kiwifruit vine decline, an emerging multifactorial disease.

Kiwifruit vine decline syndrome (KVDS) is characterized by severe root system impairment, which leads to irreversible wilting of the canopy. Plants usually collapse rapidly from the appearance of the first aboveground symptoms, without recovery even in the following seasons. The syndrome has been negatively impacting kiwifruit yield in different areas of Italy, the main producing European country, since its first outbreak in 2012. To date, a unique, common causal factor has yet to be found, and the syndrome is referred to as multifactorial. In this article, we investigated the whole biotic community (fungi, bacteria, and oomycetes) associated with the development of KVDS in three different belowground matrices/compartments (soil, rhizosphere, and root). Sampling was performed at both healthy and affected sites located in the main kiwifruit-producing area of Northwestern Italy. To address the multifactorial nature of the syndrome and to investigate the potential roles of abiotic factors in shaping these communities, a physicochemical analysis of soils was also performed. This study investigates the associations among taxonomic groups composing the microbiome and also between biotic and abiotic factors. Dysbiosis was considered as a driving event in shaping KVDS microbial communities. The results obtained from this study highlight the role of the oomycete genus Phytopythium, which resulted predominantly in the oomycete community composition of diseased matrices, though it was also present in healthy ones. Both bacterial and fungal communities resulted in a high richness of genera and were highly correlated to the sampling site and matrix, underlining the importance of multiple location sampling both geographically and spatially. The rhizosphere community associated with KVDS was driven by a dysbiotic process. In addition, analysis of the association network in the diseased rhizosphere revealed the presence of potential cross-kingdom competition for plant-derived carbon between saprobes, oomycetes, and bacteria.

Characterization and Pathogenicity of Fungal Species Associated with Dieback of Apple Trees in Northern Italy.

Severe dieback symptoms were recently observed on apple (Malus × domestica) trees in Northern Italy, representing a growing concern for producers. Surveys were conducted over a 3-year period (2019 to 2021), and five apple orchards, from 5 to 12 years old, were monitored. A total of 33 fungal isolates isolated from symptomatic plants was selected for characterization. The species identification was achieved through multilocus phylogenetic analyses performed on sequences of three genomic loci (ITS, tub2, and tef1). Morphological features were assessed, and the average growth rate at different temperatures was determined. Seven species were identified in association with dieback of apple trees: Botryosphaeria dothidea, Cadophora luteo-olivacea, Diaporthe rudis, Diplodia seriata, Eutypa lata, Kalmusia longispora, and Paraconiothyrium brasiliense. All the species were pathogenic when inoculated on healthy apple plants. B. dothidea resulted in the most aggressive infections. This study provides an insight into the fungal species diversity associated with apple dieback and provides basis for further investigations to assess the phytosanitary status of plant materials to recommend and implement effective management strategies.

Characterization of Phytopythium Species Involved in the Establishment and Development of Kiwifruit Vine Decline Syndrome.

Since 2012, the kiwifruit vine decline syndrome (KVDS) has progressively compromised Italian kiwifruit orchards. Different abiotic and biotic factors have been associated with the establishment and development of KVDS. During monitoring of orchards affected by KVDS in north-western Italy during 2016-2019, 71 Phytopythium spp. were isolated. Based on maximum likelihood concatenated phylogeny on the ITS1-5.8S-ITS2 region of the rDNA, large subunit rDNA, and cytochrome oxidase I, isolates were identified as P. vexans (52), P. litorale (10), P. chamaehyphon (7) and P. helicoides (2). Phytopythium litorale and P. helicoides are reported for the first time as agents of KVDS in Italy. To demonstrate pathogenicity and fulfil Koch's postulates, representative isolates of P. vexans, P. litorale, P. chamaehyphon and P. helicoides were inoculated in potted plants. In these trials, waterlogging was applied to stress plant with a temporary anoxia and to favour the production of infective zoospores by the oomycetes. In experiments in vitro, the four species showed the highest growth at 25-30 °C, depending on the media used. P. helicoides was able to grow also at 40 °C. The four species were able to grow in vitro at a pH ranging from 5.0 to 8.0, showing that pH had less effect on growth than temperature. The present study suggests a strong role of different species of Phytopythium in the establishment and development of KVDS. Phytopythium spp. could be favoured by the average increase in soil temperatures during summer, associated with global warming.

A Quantitative Real-Time PCR Assay for Early Detection and Quantification of Ramularia mali, an Emerging Pathogen of Apple Causing Dry Lenticel Rot.

Ramularia mali is an emerging pathogen of apple (Malus domestica) causing dry lenticel rot. No preventive measures have been adopted due to the lack of knowledge about the life cycle and epidemiology of this pathogen. In a preliminary survey to identify the agents of dry lenticel rot of apple, R. mali was constantly associated with the disease. Using isolates from this survey, a SYBR Green quantitative PCR (qPCR) assay was developed, using calmodulin as target gene, for the detection and quantification of R. mali in apple fruit. The qPCR assay was validated in terms of specificity, sensitivity, repeatability, and reproducibility following the international European and Mediterranean Plant Protection Organization standard PM 7/98. The primers amplified a region of 237 bp specific to R. mali. The specificity was validated with 20 fungal species commonly found on apple, and 36 strains of R. mali and closely related species of the R. eucalypti species complex. Positive amplifications were obtained only with DNA of R. mali and no cross-reaction was detected with the other fungal species. Sensitivity was assessed with serial dilutions of target DNA and the limit of detection was 100 fg. No influence of host DNA was observed when target DNA was diluted on the DNA of Ambrosia and Golden Delicious apple. The assay permitted to detect and quantify R. mali in symptomatic and asymptomatic fruit. The presence of R. mali on asymptomatic Ambrosia and Golden Delicious apple fruit was demonstrated, and the presence of the pathogen was reported for the first time on Jeromine, Gala, Opal, and Story Inored fruit. This assay could be useful in clarifying the life cycle of this pathogen in order to build up an effective disease management strategy. Furthermore, the early detection of the pathogen on asymptomatic apple fruit could be used to forecast the development of dry lenticel rot, supporting the packinghouse operators in deciding the storage length of apple lots.

Pathogenicity, Molecular Characterization, and Mycotoxigenic Potential of Alternaria spp. Agents of Black Spots on Fruit and Leaves of Pyrus communis in Italy.

Brown and black spots, caused by Stemphylium and Alternaria species, are important fungal diseases affecting European pear (Pyrus communis) in orchards. Both fungal genera cause similar symptoms, which could favor misidentification, but Alternaria spp. are increasingly reported due to the changing climatic conditions. In this study, Alternaria spp. were isolated from symptomatic leaves and fruits of European pear, and their pathogenicity was evaluated on pear fruits from cultivar Abate Fétel, and molecular and chemical characterization were performed. Based on maximum likelihood phylogenetic analysis, 15 of 46 isolates were identified as A. arborescens species complex (AASC), 27 as A. alternata, and four as Alternaria sp. Both species were isolated from mature fruits and leaves. In pathogenicity assays on pear fruits, all isolates reproduced the symptoms observed in the field, by both wound inoculation and direct penetration. All but one isolate produced Alternaria toxins on European pears, including tenuazonic acid and alternariol (89.1% of the isolates), alternariol monomethyl ether (89.1%), altertoxin I (80.4%), altenuene (50.0%), and tentoxin (2.2%). These isolates also produced at least two mycotoxins, and 43.5% produced four mycotoxins, with an average total concentration of the Alternaria toxins exceeding 7.58 × 106 ng/kg. Our data underline the potential risks for human health related to the high mycotoxin content found on fruits affected by black spot. This study also represents the first report of AASC as an agent of black spot on European pear in Italy.

Rapid Detection of Monilinia fructicola and Monilinia laxa on Peach and Nectarine using Loop-Mediated Isothermal Amplification.

Monilinia laxa and M. fructicola are two causal agents of brown rot, one of the most important diseases in stone fruit. Two species cause blight on blossoms and twigs and brown rot on fruit in pre- and postharvest. Both species are distributed worldwide in North and South America, Australia, and Japan. In Europe, M. laxa is endemic, while M. fructicola was introduced in 2001 and it is now widespread in several countries. Currently, both species coexist in European stone fruit orchards. Monilinia spp. overwinter in cankers and mummified fruit. Mummy monitoring during winter permits growers to understand which species of Monilinia will be prevalent in an orchard during the following season, permitting planning of an appropriate crop protection. Traditionally, the identification has been carried out using morphological features and even with polymerase chain reaction (PCR)-based assays that requires time and well-equipped laboratories. In this study, two isothermal-based methods were designed to identify these pathogens in a faster way than using traditional methods. The loop-mediated amplification (LAMP) assays were validated on some isolates of Monilinia spp. coming from the mummy monitoring according to the international European and Mediterranean Plant Protection Organization standard (PM7/98), taking into account specificity, sensitivity, repeatability, and reproducibility. The sensitivity of both assays was checked by monitoring (at different time points) two nectarine varieties artificially inoculated and stored at two different temperatures. The reliability of both LAMP assays against the quantification of the inoculum was compared with previously published quantitative PCR assays. Both LAMP methods were able to detect a low number of cells. These LAMP methods could be a useful tool for monitoring brown rot causal agents in the field and during postharvest.