First Report of Charcoal Rot Caused by Macrophomina phaseolina on Hemp (Cannabis sativa) Varieties Cultivated in Southern Spain

Abstract

In June 2015 and July 2016, charcoal rot symptoms were observed in plants of different industrial and medicinal hemp varieties grown in two different fields located in southern Spain (Los Chapatales and Alcala del Rio, Seville). In Los Chapatales, disease incidence was 22% in variety Futura 75. In Alcala del Rio, the medicinal hemp varieties Sara and Aida (disease incidence: 25.5 and 37.1%, respectively) were more susceptible than varieties Theresa (3.8%), Pilar (3.2%), and Juani (2.7%). In both fields, affected plants developed a systemic chlorosis, rapidly wilted, showed necrosis, and died. Before the stalk was completely desiccated, internal tissues appeared soft and fluffy. Discoloration of the stalk was detected near the soil line where small black sclerotia were observed. Roots were necrotic with areas of brown-violet, unprotected vascular cambium. Plant samples were collected from both locations and a fungus was consistently isolated from symptomatic stem tissues that had been surface disinfested and plated on potato dextrose agar (PDA) media. After 7 days of incubation at 30°C in the dark, all isolates produced numerous, dark, ovoidal shaped sclerotia with an average diameter of 114 µm (range: 89 to 141 µm). Based on sclerotial morphology, all isolates were identified as Macrophomina phaseolina (McPartland et al. 2000). An isolate labeled as TOR-840 (from Alcala del Rio) was characterized as susceptible to chlorate, with optimum growth at 25 to 30°C and no growth at 5 or 10°C. Pathogen identification was confirmed by amplifying and sequencing two genetic regions: TEF-1α region (GenBank accession no. MG434668) using primers EF1-728F (Carbone and Kohn 1999) and EF2 (O’Donnell et al. 1998), and CAL region (MG434669) using primers CAL-228F and CAL-737R for PCR amplification (Carbone and Kohn 1999). Sequences were compared with those in the NCBI GenBank database using a BLAST search and had 99% nucleotide sequence identity with M. phaseolina for CAL region and 100% for TEF-1α region. Inoculum for pathogenicity tests was produced by growing isolate TOR-840 in PDA media. After 1 week, six plants for each variety (Sara and Pilar, the most and the least susceptible in field trials, respectively) were inoculated by substrate irrigation of each plant with 150 ml of a sclerotia suspension (10⁴ sclerotia/ml). Plants were held at 27°C and 50/70% relative humidity (day/night) in a growth chamber with a 16-h photoperiod. Pathogenicity assays were carried out in a randomized complete block design with three blocks of two plants each. After 3 months of incubation, the mortalities of the inoculated plants were 83.3 and 16.7% for Sara and Pilar, respectively. No mortality was observed in control plants. Although Pilar had lower mortality rates than the variety Sara, M. phaseolina was reisolated from stems and petioles of 83.3% of the inoculated plants in both varieties. Charcoal rot has been reported on Cannabis sativa L. varieties in Italy, Cyprus, the United States of America, and Yugoslavia (McPartland et al. 2000). Considering that M. phaseolina has been reported in several crops in southern Spain and that effective control measures have not yet been found, susceptible hemp varieties are discouraged to be cultivated and suitable crop rotations must be ensured.