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 Honaunau, 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), ß-tubulin (ßtub), 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, ßtub, 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.

First report of bacterial leaf spot caused by Pseudomonas cichorii on Monstera adansonii in Hawai'i, USA.

Monstera adansonii is a popular ornamental house plant prized for its small size and unique leaf fenestrations. In Hawai'i, it is also sold as cut foliage (combined value ~$21K; USDA NASS 2019). In January 2022, yellow chlorotic lesions that progressed to greyish-black and, finally, to brown necrotic lesions were observed primarily along the margins of fenestrations on M. adansonii foliage at a plant nursery in Hilo, HI. All 100 variegated specimens in 4-inch pots were infected, exhibiting symptoms along the lighter yellowish-white margins. The green, unvariegated variety planted along a fence for cut foliage exhibited an infection rate of 10%. Symptomatic leaf tissue was disinfected for 1 minute in a 10% bleach solution. Tissue from the margins of leaf spots was subsequently dissected, soaked in sterile distilled water for 1 hour, and plated on Luria-Bertani (LB) agar. Plates contained nearly pure cream-colored bacterial colonies with undulate margins. Isolates were established from single colonies. One isolate (BCB001) was transferred to King's medium B (KMB) and culture fluorescence was observed under 365 nm UV light. Isolate BCB001, which was gram-negative, was identified as Pseudomonas cichorii based on the LOPAT scheme (Schaad et al. 2001). A partial 16S rRNA gene product (495 bp) using primers Y1/Y3 (Cruz et al. 2001) was sequenced and compared in GenBank (accession no. OQ875210) and was 100% identical to multiple accessions of P. cichorii in the NCBI database. Bacterial identity was further confirmed using the P. cichorii-specific primers Hrp1a/Hrp2a (Cottyn et al. 2011) to amplify and sequence a 790 bp fragment (accession no. OQ850761), which was identical to accession no. MH396007, a P. cichorii isolate recovered from Thai basil in Hawai'i. To prove pathogenicity, strain BCB001 was grown on LB agar for 48 h at 27°C and suspended in sterile water at 107-108 CFU/ml. Four healthy, 2-month-old unvariegated M. adansonii plants produced from cuttings were syringe inoculated following the protocol of Wang et al. (2022). Leaves were injected with sterile water using the same methods and acted as negative controls. Plants were placed in clear plastic bags and held at 24°C with 12 h light for 48 hours in a growth chamber, after which time the plants were removed from the bags and incubated under the same conditions for the remainder of the experiment. Leaf spots were not present on any of the control leaves or on noninjected leaves of the plants after five days of incubation. Grey to black, water-soaked leaf spots 0.84 - 15.24 mm in diameter were present on all injected leaves (96% of the injection sites) 2 days post-inoculation (DPI), which were identical to the original diseased samples. At 5 DPI, spots became dark brown to black with a yellow halo, and the affected tissue was completely collapsed. Bacterial colonies were consistently re-isolated from the lesion margins of inoculated plants and morphologically (LB and KMB) and molecularly (Hrp) identified as P. cichorii, thus fulfilling Koch's postulates. To the best of our knowledge, this is the first report of bacterial leaf spot caused by P. cichorii on M. adansonii in Hawai'i. Since M. adansonii is an ornamental plant that is prized for its leaves, leaf spots caused by P. cichorii can reduce the marketability of inventory. To avoid further spread, increasing plant spacing to improving airflow, decreasing the amount of watering, avoiding mist irrigation, and carefully removing and discarding diseased leaves are suggested.