First Report of white root rot of hemp (Cannabis sativa L.) caused by Dematophora necatrix in Campania region (Southern Italy)

Industrial hemp (Cannabis sativa L.) was cultivated in Italy until the end of the Second World War. Since then, it has been abandoned and substituted with other crops mainly due to legal restrictions and public concerns. Public legislation passed in 2016, has allowed for the production of hemp seeds, flowers and fibers (law n. 242/2016). During a 2019 survey on hemp sanitary status in the province of Naples (40°57'6"12 N, 14°22'37"56 E), hemp ‘Kompolty’ with symptoms of root rot were observed at a private farm and collected for further analysis at the phytosanitary laboratory of CREA in Caserta. Death generally occurred within 2-3 weeks after the appearance of the first symptoms, occurring on ca. 10% of plants, consisting of yellowing, canopy wilt and signs of roots covered with white mycelium and fan-like mycelium under the bark. The causal agent, was isolated from small root segments, excised from symptomatic plants, the surface was disinfected with 2% sodium hypochlorite, placed on potato dextrose agar (PDA) amended with streptomycin sulphate (100mg/L) and incubated in the dark at 25°C for 5 days. Small pieces (2-3 mm) at the edge of the resulting colonies were sub-cultured onto PDA and incubated at 25°C in the dark for one week. The mycelia from 15 isolates showed pear-shaped swellings adjacent to the septa. The conidia were aseptate, hyaline, ellipsoid to ovoid, and 3-5 × 2.5-3 µm (n=50). Based on the morphological characteristics, the fungus was identified as Rosellinia necatrix Berl. ex Prill. (Singleton et al., 1992) a fungus taxonomically revised to Dematophora necatrix R. Hartig (Wittstein et al., 2020). To confirm the identification, total DNA was extracted from five isolates using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and the ITS spacer was PCR-amplified with primers ITS1-ITS4 (White et al., 1990). The size-expected amplicons of 536 bp were purified and sequenced, the resulting sequence was trimmed and deposited in GenBank under the accession number MK937913. BLAST-n analysis revealed 98.83% nucleotide identity with some representative isolates of D. necatrix (MK888684.1; KT343972.1). To fulfill Koch’s postulates, the pathogenicity tests were carried out on fifteen 4-weeks-old potted hemp plants ‘Kompolty’. The inoculation was performed by adding 3 g of millet seeds inoculated with ten mycelial plugs, taken from the margins of a D. necatrix actively growing colony, per liter of sterile peat and perlite substrate in single pots. Moreover, ten hemp plants were inoculated with sterilized millet seed and served as negative controls. All plants were incubated at 25°C. After three weeks, inoculated plants exhibited foliar chlorosis, apical wilting, and death in two weeks, similar to what was observed in the field. Control plants did not show any symptoms. The fungus was isolated from the roots in all fifteen inoculated plants and confirmed to be D. necatrix based on morphological and molecular analysis, carried out with a second primer pair EF1-983F/ EF1-2218R targeting the transcription elongation factor 1- (Rehner and Buckley., 2005) (MW541068) that showed 99.67% nt in BLAST-n analysis. To our knowledge, this is the first report of D. necatrix infecting hemp in Europe. The farm where the problem arose has a history of cultivation for the production of apples for over 30 years. Therefore, an adaptation of D. necatrix to the new host is hypothesized. An in-depth knowledge on the diseases of hemp will be needed to relaunch hemp cultivation in this area.

Screening, identification, and colonization of fungal root endophytes against Dematophora necatrix: a ubiquitous pathogen of fruit trees

The goal of the present research was to ascertain the potential root endophytic fungi against Dematophora necatrix, Hartig, the causal pathogen of white root rot in apples; however, it has an expanded range of hosts across different fruit trees. Out of 36 endophytic fungi segregated from symptomless roots of apple, wheat, maize, marigold, cherry, and garlic plants, only 9 isolates showed inhibitory effect during preliminary screening and were further assayed under in vitro, pot, and field conditions against the white root rot pathogen/disease. Under in vitro conditions, maximum mycelial inhibition of 81.48% was obtained with the isolate Aspergillus aculeatus strain C2. Microscopic studies on interaction between fungal endophyte with hyphal tips of D. necatrix revealed various morphological abnormalities in the hyphae. In glasshouse conditions, seed treatment pursued by soil application with Crinipellis tabtim strain M8 isolate was highly effective and exhibited 93.55% disease control. Similarly, under field conditions, the overall maximum disease control was exhibited by Crinipellis tabtim strain M8 (84.95%). The most promising root endophytes that were identified rely on morphological and ITS sequence analysis. Root colonization assay was performed which revealed maximum endosphere and rhizosphere colonization with Crinipellis tabtim strain M8. Additionally, confocal microscopic illustrations of transverse sections of root cells tenanted by fungal endophytes as compared to untreated control suggested the persistence and establishment of endophytes in the endosphere of apple seedlings. These findings present the first report on colonization of apple roots by fungal root endophytes suggesting an alternative and sustainable approach for management of white root rot disease.

Soil Solarization in Established Avocado Trees for Control of Dematophora necatrix

Four field experiments on the control of Dematophora necatrix in avocado orchards affected by white root rot were conducted in the Mediterranean coastal area of southern Spain during 1991 to 1994. In the unshaded locations of solarized plots, the maximal temperatures were 35 to 42°C, depending upon the year and soil depth (15 to 60 cm). Temperature increases attributable to soil solarization ranged between 4 and 8°C in unshaded areas, whereas for shaded areas they were approximately 4°C. Inoculum recovery was decreased in root samples buried at 15 to 30 cm in unshaded locations of both solarized and unsolarized plots after 3 to 5 weeks, whereas 4 to 8 weeks of solarization were required for the elimination of the pathogen buried at depths of 45 to 60 cm. In contrast, inoculum recovery ranged from 30 to 60% for samples in shaded locations of unsolarized plots. D. necatrix was not recovered from roots of infected trees in solarized plots sampled 9 months after solarization, whereas recovery from roots in unsolarized plots was similar to levels before solarization. Soil solarization in established orchards was successful in reducing viability of inoculum buried in soil and eliminated inoculum in infected roots of live trees.