First Report of Seedling Damping-Off of industrial Hemp (Cannabis sativa L.) caused by seed-transmitted Alternaria rosae in Italy.

In the years 2020-2021 as part of the activity of the Campania region hemp fiber project, variety comparison trials were carried out on 7 hemp varieties among those relevant for bast fiber production. During the trials, in particular on the cv. Fibrante, a consistent problem was noted: a noticeable germination failure (80-90%) occurred during the emergence of seedlings. Therefore, experiments were conducted to ascertain the possible presence of seed-borne pathogens. Tests were carried out on 100 seeds that were surface disinfected with 2% sodium hypochlorite solution for 3 min, rinsed in sterile distilled water three times and dried on sterile filter paper. The seeds were plated on potato dextrose agar (PDA Oxoid™) amended with 100 mg L-1 of streptomycin sulphate, kept at 24°C in the dark and observed daily. Growing colonies were subcultured on PDA for 10 days and, subsequently, twenty purified fungal isolates were obtained by single spore isolation. Colonies of these isolates on PDA were initially grayish-white and then turned dark olive green with abundant cotton-like aerial hyphae. On potato carrot agar (PCA) medium, these isolates produced light brown and solitary conidiophore with septum. Conidia were obclavate or pyriform, brown, with 1-3 transverse septa and 0-3 longitudinal septa, and measured 12.5 to 28.5 × 5 to 15 µm (n=50). The morphological characteristics observed under the light microscope were consistent with that of Alternaria spp. (Simmons 2007). In order to characterize the representative isolate, total DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and 3 genes were PCR-amplified: the ITS spacer using the primer pair ITS1-ITS4 (White et al., 1990), the transcription elongation factor 1- using the primer pair EF1-983F/ EF1-2218R (Rehner and Buckley., 2005) and the RNA polymerase II second largest subunit (RPB2) using the primer pair RPB2-5F2/fRPB2-7cR (Sung et al 2007; Liu et al 1999). The size-expected amplicons were purified and sequenced at the BMR Genomics (Padova, Italy) and the resulting sequences were deposited in GenBank under the accession numbers ON556507, ON601003, ON601004. BLAST-n analysis revealed 98 to 99% nucleotide identity with some representative isolates of Alternaria rosae E.G. Simmons & C.F. Hill (KU375630.1, XM_046169884.1, XM_046168987.1). To fulfill Koch's postulates, 100 hemp-certified seed were disinfected as mentioned above, left to germinate on the water-agar to discard potentially infected seeds and finally sowed in sterile peat-soil mix (1:1 v/v). The inoculum consisted of 10 mL of 105 conidial suspension obtained by the representative isolate (Ar_H1). Negative control seeds were inoculated with sterile water. After 5-7 days 100% of inoculated seedlings showed weak germinative vigor with yellowing of the epicotyls and dark areas on the root. The tissue narrowed and turned necrotic with abundant white mycelium covering the entire seedling. Small pieces of necrotic roots were plated on PDA and the same Alternaria-like colonies grew in 10 days. DNA sequencing confirmed the presence of A. rosae. Alternaria spp. are fungi that produce a wide range of toxic metabolites, harmful to food safety in the food uses of the seed. This finding further highlights that the quality of the hemp seed must be considered as a priority aspect in the entire hemp supply chain. To the best of our knowledge, this is the first report of A. rosae as seed-borne fungus on hemp.

Monitoring of the copper persistence on plant leaves using pulsed thermography.

Copper-based fungicides are largely used in agriculture in the control of a wide range of plant diseases. Applied on plants, they remain deposited on leaf surfaces and are not absorbed into plant tissues. Because of accumulation problems and their ecotoxicological profiles in the soil, their use needs to be monitored and controlled, also by using modern technologies to better optimize the efficacy rendering minimum the amount of copper per season used. In this work, we test a novel approach based on pulsed thermography to evaluate the persistence of the copper on plant leaves so that the time between two applications should be the minimum needs. We monitored the thermal response observed on different treatments of both grapevine and tobacco plants over a 3-week period. Our experimental results demonstrate that the new methodological approach based on pulsed thermography can be an effective tool to evaluate in real time the presence of copper on differently treated plants allowing a tentative quantification and, therefore, to optimize its use in the agricultural practices, according also to the European Regulation n. 1107/2009.

Root and stem rot, and wilting of olive tree caused by Dematophora necatrix and associated with Emmia lacerata in Central Italy.

Lethal wilting was observed on young olive trees cv Favolosa in a grove in central Italy. White mycelial strands wrapped the basal portion of the stems that had been buried during planting. The bark was rotted and the xylem was discoloured. A fungal morphotype was strictly associated with symptomatic plants and identified as Dematophora (ex Rosellinia) necatrix. Pathogenicity tests on cvs Favolosa, Leccino and Ogliarola demonstrated that D. necatrix was the causal agent of the disease. Our investigations revealed that infections occurring during autumn and winter greatly favour the disease. By applying a marcottage to the inoculation point, we accelerated the course of the disease and mimicked the lethal outcome observed in the field. In in vitro tests, seven systemic (potential) fungicides strongly inhibited D. necatrix. Dentamet, Al-phosphite and Thiophanate methyl were selected to be tested in planta with a curative and preventive modality. Only Thiophanate methyl, in preventive modality, fully protected the plants from disease progression throughout the observation period. An additional fungal species was strictly associated with both diseased and apparently healthy plants. Morphological and molecular features identified the fungus as Emmia lacerata, a polypore species within the Irpicaceae, which is the agent of white rot on dead woody substrates. To our knowledge, this is the first time that E. lacerata has been reported in Italy and worldwide on olive trees. Inoculation of ?Favolosa' trees revealed that it colonizes the xylem without causing visible alterations. The possible role of E. lacerata in the olive tree-D. necatrix pathosystem is discussed. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10658-022-02458-1.

Potential Use of Cardunculus Biomass on Pleurotus eryngii Production: Heteroglycans Content and Nutritional Properties (Preliminary Results).

The new perspective of using waste biomass to cultivate mushrooms as a source of protein for human nutrition, in line with the circular economy principles, is receiving increasing attention in the scientific community and represents great wealth in terms of environmental sustainability. Pleurotus eryngii is a mushroom also known as cardunculus mushroom due to its ability to grow on this plant. This study explores the potential intrinsic properties of cardunculus (for example, the presence of inulin in the roots) as raw material for the growth of cardunculus mushrooms, and the influence on heteroglycan content and nutrition parameters of the fruiting bodies. Both mycelium and fruiting bodies were used to determine the heteroglycan content in the presence of inulin or cardunculus roots rich in inulin. To produce heteroglycans from P. eryngii in greater quantities and shorter times without having to wait for the formation of the fruiting bodies, the mycelium could be used. The results showed that the presence of cardunculus biomass positively influences the heteroglycan content of P. eryngii. In terms of nutritional parameters, higher contents of polyphenols, flavonoids, anthocyanins, and antioxidant activity were detected in P. eryngii grown on the cardunculus stem and root substrate. In conclusion, recycling cardunculus biomass to generate growth blocks for edible mushrooms is a winning choice due to the opportunity to use this biomass waste, which is gaining more and more attention due to the increase in cultivated areas and the use of fruiting bodies of P. eryngii as a functional food and source of molecules with potential biological activities.

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.

An overview of the current plant biostimulant legislations in different European Member States.

Plant biostimulants are borderline substances that play an intermediate role between plant protection products and fertilisers. At present, such substances are regulated by national laws and have different names in the various European Member States. The aim of this paper is to provide an overview on the activity of these substances and on the national laws that regulate them, as they vary considerably from one Member State to another. The greatest difficulty in terms of the correct regulatory framework for these substances is related to their heterogeneity. This situation creates uncertainties for operators, control authorities and bodies that certify and control the organic production, and strongly limits the growth of these substances. This problem will be overcome with the amendment of Regulation EC No 2003/2003 which will shortly extend its scope to the category of plant biostimulants included within the fertilising additives.