The native ‘ōhi’a lehua (Metrosideros polymorpha) has cultural, biological and ecological significance to Hawai’i, but it is seriously threatened by a disease commonly referred to as rapid ‘ōhi’a death (ROD). Preliminary investigations showed that a Ceratocystis species similar to C. fimbriata s.lat. was the cause of the disease. In this study, we used a combination of the phylogenetic, morphological and biological species concepts, as well as pathogenicity tests and microsatellite analyses, to characterise isolates collected from diseased ‘ōhi’a trees across Hawai’i Island. Two distinct lineages, representing new species of Ceratocystis, were evident based on multigene phylogenetic analyses. These are described here as C. lukuohia and C. huliohia. Ceratocystis lukuohia forms part of the Latin American clade (LAC) and was most closely associated with isolates from Syngonium and Xanthosoma from the Caribbean and elsewhere, including Hawai’i, and C. platani, which is native to eastern USA. Ceratocystis huliohia resides in the Asian-Australian clade (AAC) and is most closely related to C. uchidae, C. changhui and C. cercfabiensis, which are thought to be native to Asia. Morphology and interfertility tests support the delineation of these two new species and pathogenicity tests show that both species are aggressive pathogens on seedlings of M. polymorpha. Characterisation of isolates using microsatellite markers suggest that both species are clonal and likely represent recently-introduced strains. Intensive research is underway to develop rapid screening protocols for early detection of the pathogens and management strategies in an attempt to prevent the spread of the pathogens to the other islands of Hawai’i, which are currently disease free.

Myrtaceae (myrtle family) 'ōhi'a, 'ōhi'a lehua, lehua (Hawai'i)

Miconia calvescens (Melastomataceae), from the Neotropics, is a noxious forest weed in Hawaii. We evaluated an isolate of Colletotrichum gloeosporioides that causes leaf spots on Miconia spp. in Brazil for its potential in biological control. Hawaii has no native Melastomataceae genera but does have members of 12 introduced genera. Following Wapshere's centrifugal phylogenetic method (2), eight species of Melastomataceae genera in Hawaii were inoculated in addition to Miconia spp. Naturalized and native Hawaiian members of the order Myrtales also were inoculated to determine host specificity, including Terminalia catappa (Combretaceae); Cuphea hysopifolia and C. ignea (Lythraceae); Arthrostema ciliatum, Clidemia hirta, Dissotis rotundifolia, Heterocentron subtriplinervium, Medinilla scortechenii, Melastoma candidum, Pterolepsis glomerata, and Tibouchina herbaceae (Melastomataceae); Eucalyptus grandis, Eucalyptus microcorys, Eugenia reinwardtiana, Eugenia uniflora, Leptospermum laevigatum, Melaleuca quinquenervia, Metrosideros polymorpha, Psidium guajava, and Syzgium malaccanse (Myrtaceae); Fuchsia magellanica and Oenothera stricta (Onagraceae); and Wikstroemia oahuensis and W. uva-ursi (Thymelaeaceae). All M. calvescens plants were grown from seed collected in Hawaii. Other test plants were grown from seeds or cuttings in artificial potting medium in a greenhouse. Plants had 6 to 8 mature leaves when inoculated. C. gloeosporioides was cultured on 10% potato dextrose agar supplemented with plain agar (35 g/liter) and incubated under constant fluorescent illumination at 20°C. Conidia were harvested by flooding 10-to 14-day-old cultures with sterile tap water, followed by light scraping with a scalpel. Conidial suspensions were adjusted to 106 conidia per ml and applied to both leaf surfaces with a hand-held sprayer. Inoculated plants were kept at 100% relative humidity and 16 to 25°C for 48 h. Four replicate plants and one plant of M. calvescens per species were inoculated. Plants were observed for symptom development for up to 6 weeks. The entire test was repeated once. Lesions were visible after 7 to 10 days. Young lesions had chlorotic halos and expanded in a roughly circular pattern to diameters of 5 to 10 mm. Mature lesions developed necrotic centers, coalesced, and became dry and brittle with age, resulting in extensive leaf necrosis. Defoliation of moderately to severely infected leaves occurred ≈ 30 days after inoculation. With the exception of M. calvescens, C. gloeosporioides did not produce visible symptoms on test plants. The failure of Clidemia hirta, the taxonomic species most closely related to M. calvescens, to become symptomatic was particularly significant relative to the centrifugal phylogenetic concept. The results demonstrate that our pathogen (VIC 19306) is distinct from C. gloeosporioides f. sp. clidemiae (1), which did not infect M. calvescens. We designate our pathogen C. gloeosporioides f. sp. miconiae. Voucher specimens (VIC 19306, Sana, RJ, 24.II.1998, and R. W. Barreto) and cultures are maintained at the Departamento de Fitopatologia, Universidade Federal de Viçosa MG, Brazil. References: (1) E. E. Trujillo et al. Plant Dis. 70:974, 1986. (2) A. J. Wapshere. Ann. Appl. Biol. 77:201, 1974.

Coffee is one of the most economically valuable specialty crops for which Hawaii is famous. It is produced commercially on >6,900 acres across six islands by more than 1,470 growers. It has a raw crop value of $55.9 million, while the value-added benefits of coffee-related industries exceed $148.5 million (USDA, 2021). In addition to high product quality, Hawaii also has the distinction of being the last major coffee growing region that is free of coffee leaf rust (CLR), a highly damaging disease caused by the obligate parasitic fungus Hemileia vastatrix Berk. & Broome. On October 21, 2020, a coffee grower in Maui County, Hawaii reported the presence of coffee leaf rust-like symptoms on coffee plants (var. ‘Typica’) at their farm with many trees heavily defoliated. Foliar symptoms consisted of yellowish-orange, circular lesions that often coalesced. On abaxial surfaces, these lesions appeared powdery. Urediniospores were mostly reniform, (25)27-34(36) × (17)18-28(29) µm, strongly echinulated on the upper (convex) surface and smooth on the lower (concave) surface, with hyaline to pale yellow-orange walls. Urediniospores incubated in molecular grade water for 10 min at 95 oC served as template for PCR targeting the internal transcribed spacer region of fungi using primers ITS1/ITS2, ITS3/ITS4, and ITS1/ITS4 (White et al., 1990). Amplification products underwent direct Sanger-based sequencing. Following primer sequence trimming, the sequence reads were assembled using CAP3 (Huang and Madan, 1999) and deposited in GenBank (Accession MW228837). Blastn analysis revealed > 99% nucleotide identity with isolates of H. vastatrix from Mexico (eg. KX260251) and Brazil (eg. MF627828). A voucher material from which both the morphological and molecular assays were performed was deposited in the National Fungus Collection (BPI 924818). Subsequent surveys on the islands of Hawaii, Lanai, and Oahu revealed the presence of coffee plants with symptoms of coffee leaf rust. To confirm pathogenicity, urediniospores from a symptomatic plant growing in Holualoa, Hawaii, were collected in gelatin capsules using a G-R Electric Manufacturing Portable Vacuum Pump with a mini cyclone spore adapter. The concentration of spores was adjusted to 1 x 105 spores/ml in sterile water using a Brightline Hemocytometer. The spore suspension was brushed onto the abaxial side of leaves from two C. arabica var. ‘Typica’ plants using a camel hair paintbrush. Two control plants were mock-inoculated with sterile water. Plants were placed in a dark humid chamber set at 22 oC for 48 hours, after which it was adjusted to 12 hours light under cool white fluorescent lighting. After 20 days small, chlorotic spots were visible on the adaxial leaf surface and a few spots contained orange urediniospores on the abaxial surface. Lesions expanded by day 34, followed by necrosis of the center area of several leafspots and leaf abscission was observed at day 70. The experiment was conducted twice. H. vastatrix was confirmed by both morphological and molecular examination of urediniospores recovered from the observed lesions. All mock-inoculated control plants remained asymptomatic. To our knowledge, this is the first report of CLR on coffee in Hawaii. This discovery is of great concern since CLR threatens not only the yield and quality of Hawaii-grown coffee, but also the economic viability of this historic and culturally important industry. Statewide monitoring for CLR continues and further work is needed to mitigate the impact of this discovery through the development of short- and long-term management strategies.