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.
Kiwifruit Vine Decline Syndrome (KVDS) is a serious soil-borne disease that degrades the fine roots of both Actinidia chinensis var. deliciosa and var. chinensis. The disease seems to be the result of an interaction between several soil-borne pathogens, mostly oomycetes, and waterlogging. This work investigates the pathogenicity of the oomycete Phytopythium chamaehyphon recently isolated from roots of diseased plants. Pathogenicity was tested in 6-month-old and 1-year-old plants that after inoculation were flooded up to three times to induce symptom appearance. Leaf wilting and root rot typical of KVDS was observed in all the plants inoculated with P. chamaehyphon strain KD-15 (PCHA) and in all the positive controls potted in a mix of peat and soils collected in KVDS-affected orchards, while negative controls remained symptomless. Disease development on 6-months-old plants was characterized by unusual degradation of the not-lignified collar, occurring even in absence of flooding. Conversely, on 1-year-old plants, symptoms faithfully reproduced KVDS dynamics observed in orchard. This work confirmed the pathogenicity of P. chamaehyphon and raised new questions about the actual role of waterlogging in KVDS etiology.
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. littorale (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. littorale, 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.
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