First Report of Charcoal Rot Caused by Macrophomina phaseolina on Hemp in Croatia.

The production of industrial hemp (Cannabis sativa) has had a sharp increase in the past five years in Croatia (Mihelcic 2017). Production has been in constant increase, from 1560 ha in 2015 to 2476 ha in 2019 (PAAFRD). In August 2019, numerous (~1.5%) premature wilted hemp plants (cultivar Fibranova) were observed in commercial fields in Vladislavci (45.4646950° N, 18.5674770° E), around Osijek, Croatia. Diseased plants showed symptoms of chlorosis, rapidly wilting, necrosis and prematurely dying. The stalk of diseased plants was completely desiccated, while internal tissues were spongy and fluffy. Near the soil line, discoloration of the stalk with small spherical to oblong black microsclerotia was detected. Roots were necrotic with brown-gray areas. Twenty root and crown segments of the infected plants (2- to 3-mm long pieces) were surface sterilized with 2% NaOCl for 5 min, rinsed three times with sterile distilled water, and plated on potato dextrose agar (PDA, pH 6.2) media, containing 250 mg per liter of chloramphenicol to inhibit bacterial growth (Zveibil and Freeman 2005). The culture plates were incubated at 28 ± 2°C for 7 days in the dark and purified to obtain a pure culture that produces numerous, dark, hard, ovoidal-shaped sclerotia, averaging 140 x 52 μm (n=50). The single sclerotium isolate produced flat light to dark gray colonies with entire margins. Based on field symptoms, colony and microsclerotia morphology, the fungus was identified as Macrophomina phaseolina (Tassi) Goid (Marquez et al. 2021). Total DNA from the isolate was extracted with Extract-N-Amp Plant PCR Kit (Sigma-Aldrich Co., USA). To confirm morphological identification, part of the TEF 1-alpha gene region was amplified using EF1-728F (Carbone and Kohn 1999) and EF2 (O'Donell et al. 1998). The sequence of the isolate MP1 (212 bp - GenBank accession no. OQ389757), showed 100% nucleotide sequence identity to the reference sequence of M. phaseolina GenBank sequence MG434668 (Casano et al. 2018). Eighteen hemp plants (cv. Fibranova) were sown in six plastic pots (three hemp plants per pot) for the pathogenicity test. Ten-day-old M. phaseolina culture (isolate MP1) was used for inoculum preparation. Each pot of one-week-old plant was irrigated with 100 ml of a microsclerotia suspension (105 microsclerotia/ml)(Abied et al. 2018). Plants were held at 28°C and 70% relative humidity in a growth chamber (aralab, Fitoclima 10.000 HP) with a 16-hour photoperiod. Pots with control plants were irrigated with the same amount of sterile distilled water. Ten weeks after inoculation percentage of wilted plants was 77.78%. In the control variant all plants were healthy. M. phaseolina was reisolated from inoculated plants and morphologically identified. With the liberalization of the law, hemp production in Croatia is increasing, which could result in general disease problems and the disease caused by M. phaseolina. Charcoal rot will be expressed in years with dry and warm summers (Lodha and Mawar 2020), and relatively short, cool, rainy winters (Nevo et al. 2012), which has become common in the last decade in Croatia. To our knowledge, this is the first report of M. phaseolina on hemp in Croatia. The authors declare no conflict of interest.