First report of leaf spot caused by Paramyrothecium roridum on Coffea arabica in Hawai'i, USA.

During the 2022-2023 season, the harvested coffee crop in Hawai'i (Coffea arabica) was valued at $57.1 million (USDA NASS 2023). In September 2022, coffee leaf samples with foliar leaf spots affecting the Kona Typica variety were collected from Hōnaunau, Hawai'i, incidence <10%. The symptoms were circular, necrotic leaf spots with yellow margins, which merged, resulting in complete leaf blade coverage and subsequent leaf drop. Sporodochia were present on the abaxial leaf surface. Symptomatic leaf tissue was disinfected in 10% bleach solution for 60 seconds and chlorotic leaf tissue from the spot margins were excised and placed onto water agar and potato dextrose agar (PDA; Difco, USA). After a 7-day incubation period, pure cultures with white aerial mycelium having sporodochia arranged in concentric rings with olivaceous to black conidial masses were isolated. The conidia were aseptate, hyaline, smooth, cylindrical with rounded ends, measuring 5.1 to 6.8 μm long and 1.7 to 2.3 μm wide (n=50). Based on symptomology and cultural/morphological characteristics (Huaman-Pilco et al. 2023; Lombard et al. 2016; Pelayo-Sanchez et al. 2017), the isolates were initially identified as Paramyrothecium roridum (Tode) L. Lombard & Crous, comb. nov. (syn. Myrothecium roridum Tode). Fungal identification of isolate P22-81-2 was further confirmed using BLAST analysis of bulk sequenced PCR products of the ribosomal DNA internal transcribed spacer (ITS) region (White et al. 1990), β-tubulin (βtub), RNA polymerase II (RPB2), and calmodulin genes (Lombard et al., 2016; Huaman-Pilco et al., 2023). The gene sequences (GenBank accession nos. PP211198, PQ192517-19) were >98.4% identical to the P. roridum type specimen (CBS 357.89). A multilocus maximum likelihood phylogenetic analysis incorporating sequence data from previous relevant studies (Lombard et al., 2016; Pinruan et al. 2022) confirmed species identification. To prove pathogenicity, four, 26-month-old Kona Typica variety seedlings were foliar inoculated with a 1 X 10⁶ conidia/ml suspension using a perfume atomizer. An additional four plants were inoculated in a similar manner with sterile water which served as controls. All plants were sprayed to drip on both the upper and lower leaf surfaces and incubated in a clear plastic bag to keep the humidity levels between 90 to 100% for 48 hours at 24°C. After 48 hours, the plants were removed from the bags, placed on a greenhouse bench, and observed weekly for symptom development. Within seven days light brown sunken spots had developed on all inoculated plants. The spots continued to enlarge having a dark distinct margin, light tan center, chlorotic halo, and formed concentric rings, which were identical to the original diseased samples. Leaf spots were not present on any of the control plants. The test was conducted twice. A fungus was consistently reisolated from the leaf spot margins of inoculated plants and morphologically (PDA) and molecularly (ITS, βtub, RPB2, calmodulin) identified as P. roridum, thus fulfilling Koch's Postulates. To the best of our knowledge, this is the first report of P. roridum causing leafspots on C. arabica plants in Hawai'i. This pathogen has been reported on coffee in other parts of the world including Colombia, Costa Rica, Guatemala, Puerto Rico, and Mexico (USDA Fungus-Host Database). Under the right conditions, P. roridum has the potential to cause leafspots and defoliation resulting in economic losses for coffee growers in Hawai'i.