Vacuum Steam Treatment of Metrosideros polymorpha Logs for Eradication of Ceratocystis huliohia and C. lukuohia.

A new and devastating disease, rapid ohia death (ROD), in Hawaii led to a state quarantine that regulates interisland transport of ohia wood and plant material to prevent spread of the causal pathogens. Heat treatments of ohia logs in commercial trade were considered for phytosanitary treatment. Vacuum steam (VS) was evaluated for its ability to eradicate the pathogens, Ceratocystis lukuohia and C. huliohia, in main stem logs from ROD-affected forest trees. Replicate loads of three debarked logs (24 to 43 cm in diameter, 1.7 to 2.0 m long) were VS treated at 56°C for 30 min (five loads) or 60°C for 60 min (four loads) at a sapwood depth equal to 70% of log radius. Percentage isolation of Ceratocystis from VS and ambient temperature logs before treatment and summarized by source tree ranged from 12 to 66% and 6 to 31% based on carrot baiting assays of tissue taken from outer and inner sapwood, respectively. No viable Ceratocystis was detected in sapwood locations for the 60°C/60 min schedule or inner locations for the 56°C/30 min schedule after treatment. Only one subsample (0.48%, n = 208) of the latter schedule treatment yielded Ceratocystis. Time needed for treatment ranged from 7.4 to 15 h for the 56°C/30 min schedule and from 8.6 to 19.2 h for the 60°C/60 min schedule. These results demonstrate that VS is an effective and efficient method for treating large-diameter ohia logs that mill owners and regulatory plant pathologists may consider for use in Hawaii.

Genetic diversity among naturally-occurring strains of Beauveria bassiana associated with the introduced coffee berry borer, Hypothenemus hampei, (Coleoptera: Curculionidae) on Hawai'i Island.

The coffee berry borer (CBB), Hypothenemus hampei, is considered the most important insect pest of coffee worldwide. CBB was discovered on Hawai'i Island in 2010 and soon thereafter on the islands of O'ahu (2014) and Maui (2016). As part of an areawide effort to manage CBB in Hawai'i, we conducted a survey of naturally-occurring Beauveria associated with the beetle to complement field efficacy studies of the commercial B. bassiana strain GHA. Sampling of CBB from coffee farms or unmanaged sites in various districts on the islands of Hawai'i and O'ahu, and also from Puerto Rico, resulted in >1800 Beauveria isolates. These were initially characterized using colony morphology to differentiate strain GHA, registered for use in Hawai'i, from indigenous congenerics. A total of 114 isolates representative of these indigenous morphotypes were selected for further characterization. Sequencing of the intergenic regions B locus and EFutr identified all as Beauveria bassiana sensu stricto. Sixteen haplotypes were observed, with one more common haplotype present in 12 of 16 sites sampled on Hawai'i Island. This B locus-EFutr haplotype, designated Bb1, was the only haplotype observed in 2016 epizootics on two high-elevation coffee farms on Hawai'i Island with no history of GHA application. Many of the haplotypes showed genetic similarity to those collected from CBB from other countries, including Brazil, Columbia, Nicaragua, and Kenya, but a few were identical to those from other insect species collected in Hawai'i before 2010. This diversity suggests a mixed lineage among B. bassiana strains associated with CBB in the three Hawaiian islands.

Pathogenicity, Symptom Development, and Colonization of Metrosideros polymorpha by Ceratocystis lukuohia.

Extensive mortality of Metrosideros polymorpha (`ohi`a) trees has been associated with Ceratocystis spp. on Hawai`i Island and was named rapid `ohi`a death (ROD). Both C. lukuohia and C. huliohia have been associated with ROD, although C. lukuohia appears to be the more important pathogen. Crown observations and dissections of forest trees either wound-inoculated with, or naturally infected by, C. lukuohia were conducted to confirm pathogenicity and document patterns of host colonization. In pathogenicity trials, one of three and two of three trees inoculated with the fungus in February and August, respectively, exhibited crown wilt symptoms at 92 and 69 days after inoculation. Extensive, radial, black staining of the sapwood was found in main stems, while no crown wilt or xylem staining was found in control trees. Xylem staining, necrotic phloem, and fungus presence was noted in six trees inoculated in May to June and harvested 37 to 42 days later, and these observations were compared with those in two naturally infected trees felled in early August. Contiguous xylem staining was found in the main stems and into crowns of all diseased trees, while discontinuous streaks of xylem staining extended into the main forks and side branches. Necrotic phloem associated with xylem staining occurred on the lower stems of inoculated trees. Aside from the necrotic phloem and radial staining of the sapwood, symptom development in `ohi`a infected with C. lukuohia is similar to other systemic wilt diseases on hardwood trees. We propose Ceratocystis wilt of `ohi`a as the official name of the disease.

Real-Time PCR Assays to Detect and Distinguish the Rapid 'Ohi'a Death Pathogens Ceratocystis lukuohia and C. huliohia.

Ceratocystis lukuohia and C. huliohia are recently described fungal species that cause rapid 'ohi'a death (ROD) of Metrosideros polymorpha, Hawaii's most abundant and ecologically important native species. Although the pathogens are now widespread on Hawai'i Island, a major effort is underway to study and manage affected forests, and particularly to prevent the disease from spreading to other islands in the State or throughout the Pacific. Rapid and accurate detection is critical. Molecular diagnostic real-time PCR protocols were developed to detect and distinguish the two pathogens, suitable for detection of fungal DNA from extracts of wood, soil, and insect frass. The assays detect as few as 2 to 4 or 16 spores of C. huliohia or C. lukuohia, respectively. These assays are valuable tools for monitoring disease spread and offer a significant advantage over culture-based methods for diagnostics, requiring <1 day to arrive at definitive results.

Survey of potential fungal antagonists of Coffee Leaf Rust (Hemileia vastatrix) on Coffea arabica in Hawai'i, USA.

Hemileia vastatrix, causal agent of coffee leaf rust (CLR), is an aggressive pathogen of coffee plants worldwide. Conventional fungicides play a major role in the suppression of this disease, but a recent shift toward eco-friendly farming practices has occurred and additional novel, effective, and sustainable strategies for CLR control are needed. Naturally occurring fungal antagonists could be well-positioned to meet this demand, but these fungi need to be isolated and tested for efficacy to identify organisms with potential. In this study, a survey of fungi associated with CLR lesions in four districts of Hawai'i Island, HI, USA (Kona, Ka'u, Hamakua, and Hilo) was conducted. Coffee leaves infected with CLR were collected from 22 locations and over 600 lesions were plated on ½ APDA and CTC 4T media. DNA was extracted from purified isolates and the internal transcribed spacer region (ITS) was sequenced and analyzed by BLASTn. In total, 194 isolates comprising 50 taxa were recovered. Several of the genera are known antagonists of CLR or other plant pathogens, including Simplicillium, Akanthomyces, Cladosporium, Fusarium, and Clonostachys. The wide diversity of fungi associated with CLR lesions provide a wealth of possibilities for identifying potential CLR antagonists that could serve as a valuable tool for coffee farmers as part of an integrated pest management plan.

Screening promoters for Anthurium transformation using transient expression.

KEY MESSAGE : There are multiple publications on Anthurium transformation, yet a commercial product has not been achieved. This may be due to use of non-optimum promoters here we address this problem. Different promoters and tissue types were evaluated for transient ß-glucuronidase (GUS) expression in Anthurium andraeanum Hort. 'Marian Seefurth' following microprojectile bombardment. Plasmids containing the Ubiquitin 2, Actin 1, Cytochrome C1 from rice, Ubiquitin 1 from maize and 35S promoter from Cauliflower Mosaic Virus fused to a GUS reporter gene were bombarded into in vitro grown anthurium lamina, somatic embryos and roots. The number of GUS foci and the intensity of GUS expression were evaluated for each construct. Ubiquitin promoters from rice and maize resulted in the highest number of expressing cells in all tissues examined. Due to the slow growth of anthurium plants, development of transgenic anthurium plants takes years. This research has rapidly identified multiple promoters that express in various anthurium tissues facilitating the development of transformation vectors for the expression of desirable traits in anthurium plants.

Coffee Leaf Rust (Hemileia vastatrix) from the Recent Invasion into Hawaii Shares a Genotypic Relationship with Latin American Populations.

Hawaii has long been one of the last coffee-producing regions of the world free of coffee leaf rust (CLR) disease, which is caused by the biotrophic fungus Hemileia vastatrix. However, CLR was detected in coffee farms and feral coffee on the island of Maui in February 2020 and subsequently on other islands of the Hawaiian archipelago. The source of the outbreak in Hawaii is not known, and CLR could have entered Hawaii from more than 50 coffee-producing nations that harbor the pathogen. To determine the source(s) of the Hawaii inoculum, we analyzed a set of eleven simple sequence repeat markers (SSRs) generated from Hawaii isolates within a dataset of 434 CLR isolates collected from 17 countries spanning both old and new world populations, and then conducted a minimum spanning network (MSN) analysis to trace the most likely pathway that H. vastatrix could have taken to Hawaii. Forty-two multilocus genotypes (MLGs) of H. vastatrix were found in the global dataset, with all isolates from Hawaii assignable to MLG 10 or derived from it. MLG 10 is widespread in Central America and Jamaica, making this region the most probable source of inoculum for the outbreak in Hawaii. An examination of global weather patterns during the months preceding the introduction of CLR makes it unlikely that the pathogen was windborne to the islands. Likely scenarios for the introduction of CLR to Hawaii are the accidental introduction of spores or infected plant material by travelers or seasonal workers, or improperly fumigated coffee shipments originating from Central America or the Caribbean islands.

Canker and Twig Dieback of Blueberry Caused by Pestalotiopsis spp. and a Truncatella sp. in Chile.

Blueberry (Vaccinium spp.) has great economic importance in Chile, which currently has about 8,500 ha being cultivated. Recently, the presence of canker and dieback symptoms has been observed along the productive blueberry zone of Chile. Species of Pestalotiopsis and Truncatella were consistently isolated from diseased samples in 22 different locations. Therefore, the objective of this study was to identify and characterize the species of Pestalotiopsis and Truncatella associated with canker and twig dieback symptoms on blueberry. Forty-nine isolates were obtained on acidified potato dextrose agar in 2006 and 2007. These isolates were identified as Pestalotiopsis clavispora, P. neglecta, and Truncatella (=Pestalotia) angustata on the basis of colony characteristics and conidial morphology. This identification was verified by internal transcribed spacer analysis of DNA. Isolates of P. clavispora, P. neglecta, and T. angustata were pathogenic on apple, kiwifruit, and blueberry fruit. Similarly, isolates of P. clavispora were pathogenic on detached blueberry twigs of cv. O'Neal. Additionally, three selected isolates of P. clavispora induced light-brown canker lesions, surrounded by a reddish halo, and shoot dieback after twig inoculations on 2-year-old twigs of blueberry cvs. O'Neal, Bluecrop, Brightwell, Brigitta, Duke, Elliot, and Misty. Among blueberry cultivars, Brightwell and O'Neal were the most susceptible and Bluecrop and Misty the least susceptible, while Elliot, Brigitta, and Duke were moderately susceptible to P. clavispora. These pathogens were isolated consistently from inoculated plants, confirming Koch's postulates. P. clavispora was highly sensitive to fludioxonil and pyraclostrobin with a median effective concentration of 0.06 to 0.08 and 0.04 to 0.8 µg/ml, respectively. Therefore, the results of this study indicate that P. clavispora, P. neglecta, and T. angustata are primary pathogens that can cause canker lesions and dieback symptoms on blueberry not previously described in Chile. However, these results do not exclude that other species of these genera or other plant-pathogenic fungi (e.g., Botryosphaeria, Pestalotia, and Phomopsis spp.) may eventually be involved in this syndrome of blueberry.

Organ-specific transcriptome profiling of metabolic and pigment biosynthesis pathways in the floral ornamental progenitor species Anthurium amnicola Dressler.

Anthurium amnicola Dressler possesses a number of desirable and novel ornamental traits such as a purple-colored upright spathe, profuse flowering, and floral scent, some of which have been introgressed into modern Anthurium cultivars. As a first step in identifying genes associated with these traits, the transcriptome from root, leaf, spathe, and spadix from an accession of A. amnicola was assembled, resulting in 28,019 putative transcripts representing 19,458 unigenes. Genes involved in pigmentation, including those for the metabolism of chlorophyll and the biosynthesis of carotenoids, phenylpropanoids, and flavonoids were identified. The expression levels of one MYB transcription factor was highly correlated with naringenin 3-dioxygenase (F3H) and dihydroflavonol-4-reductase (DFR) in leaves, whereas a bHLH transcription factor was highly correlated with flavonoid 3'-monooxygenase (F3'H) and a DFR in spathes, suggesting that these two transcription factors might regulate flavonoid and anthocyanin synthesis in A. amnicola. Gene sequence and expression data from four major organs of A. amnicola provide novel basal information for understanding the genetic bases of ornamental traits and the determinants and evolution of form and function in the Araceae.

Identification and Characterization of Pestalotiopsis spp. Causing Scab Disease of Guava, Psidium guajava, in Hawaii.

Guava is one of the most widely grown plants in the tropics; however, it is affected by many fruit rot diseases. Fruit diseases decrease the marketability of fresh fruit and fruit for processing. A survey of scab disease was conducted at the USDA/ARS Tropical Plant Genetic Resource Management Unit in Hilo, HI, where more than 50 accessions of guava are grown. Symptoms observed were gray/light brown lesions surrounded by dark brown borders on leaves and brown, raised, corky, necrotic lesions on the exocarp of fruit which progressed as the fruits matured. Seventeen isolates from infected fruit, six isolates from lesions on leaves, and nine isolates from additional crops surrounding the guava trees were collected. The main fungi consistently isolated from symptomatic leaves and fruit were Pestalotiopsis spp. Morphology, colony characteristics, and pathogenicity of the isolates were examined and potential sources of host resistance were identified for germplasm characterization studies. Molecular methods were used to identify four Pestalotiopsis taxa (P. clavispora, P. microspora, P. sp. GJ-1, and P. disseminata) on guava in Hawaii. To our knowledge, this is the first report of traditional and molecular methods of identification and characterization being used for fungal pathogens of guava in Hawaii.

Isolation and Characterization of Burkholderia gladioli from Orchids in Hawaii.

Bacterial diseases of orchids continue to be serious problems. Bacterial strains were isolated from orchid plants exhibiting disease symptoms in Hawaii. Small to large leaf spots with or without water-soaking or soft rots were observed on various orchid genera, including Dendrobium, Oncidium, and Miltonia spp. and hybrids. Bacteria isolated and cultured from the lesions were tentatively identified using analytical profile index (API) strips and standard physiological and biochemical tests, and confirmed by species-specific polymerase chain reaction and sequencing of the 16S rRNA gene. The variation in pathogenic, morphological, cultural, and molecular characteristics of the orchid isolates also was evaluated. In our studies, a gramnegative, aerobic, rod-shaped bacterium that produced pale yellow, opaque, round colonies with entire margins on nutrient broth yeast extract agar (NBY) was isolated consistently from diseased orchid plants. On yeast dextrose calcium carbonate agar, the isolates produced brownishyellow, nonmucoid colonies, with the majority of the strains secreting a diffusible yellow or tan pigment into the media. The bacterium was identified as Burkholderia gladioli. Molecular analysis indicated very little diversity in the 16S rDNA gene. Testing B. gladioli isolates using media containing copper or streptomycin indicated varying levels of resistance (copper resistant = Cur; streptomycin resistant, Smr), with approximately 75% of the strains resistant to copper and 94% of the strains resistant to streptomycin. The minimum inhibitory concentration (MIC) of cupric sulfate among Cur strains ranged from 50 to 1,000 µg/ml and the MIC of streptomycin was 50 to 100 µg/ml for all Smr B. gladioli strains tested. Field and laboratory data suggest the frequent use of these chemicals in nurseries may have inadvertently resulted in the development of copper and streptomycin resistance in B. gladioli from orchids.

The algT gene of Pseudomonas syringae pv. glycinea and new insights into the transcriptional organization of the algT-muc gene cluster.

The phytopathogenic bacterium Pseudomonas syringae pv. glycinea infects soybean plants and causes bacterial blight. In addition to P. syringae, the human pathogen Pseudomonas aeruginosa and the soil bacterium Azotobacter vinelandii produce the exopolysaccharide alginate, a copolymer of d-mannuronic and l-guluronic acids. Alginate production in P. syringae has been associated with increased fitness and virulence in planta. Alginate biosynthesis is tightly controlled by proteins encoded by the algT-muc regulatory gene cluster in P. aeruginosa and A. vinelandii. These genes encode the alternative sigma factor AlgT (sigma(22)), its anti-sigma factors MucA and MucB, MucC, a protein with a controversial function that is absent in P. syringae, and MucD, a periplasmic serine protease and homolog of HtrA in Escherichia coli. We compared an alginate-deficient algT mutant of P. syringae pv. glycinea with an alginate-producing derivative in which algT is intact. The alginate-producing derivative grew significantly slower in vitro growth but showed increased epiphytic fitness and better symptom development in planta. Evaluation of expression levels for algT, mucA, mucB, mucD, and algD, which encodes an alginate biosynthesis gene, showed that mucD transcription is not dependent on AlgT in P. syringae in vitro. Promoter mapping using primer extension experiments confirmed this finding. Results of reverse transcription-PCR demonstrated that algT, mucA, and mucB are cotranscribed as an operon in P. syringae. Northern blot analysis revealed that mucD was expressed as a 1.75-kb monocistronic mRNA in P. syringae.

Alginate gene expression by Pseudomonas syringae pv. tomato DC3000 in host and non-host plants.

Pseudomonas syringae produces the exopolysaccharide alginate, a copolymer of mannuronic and guluronic acid. Although alginate has been isolated from plants infected by P. syringae, the signals and timing of alginate gene expression in planta have not been described. In this study, an algD : : uidA transcriptional fusion, designated pDCalgDP, was constructed and used to monitor alginate gene expression in host and non-host plants inoculated with P. syringae pv. tomato DC3000. When leaves of susceptible collard plants were spray-inoculated with DC3000(pDCalgDP), algD was activated within 72 h post-inoculation (p.i.) and was associated with the development of water-soaked lesions. In leaves of the susceptible tomato cv. Rio Grande-PtoS, algD activity was lower than in collard and was not associated with water-soaking. The expression of algD was also monitored in leaves of tomato cv. Rio Grande-PtoR, which is resistant to P. syringae pv. tomato DC3000. Within 12 h p.i., a microscopic hypersensitive response (micro-HR) was observed in Rio Grande-PtoR leaves spray-inoculated with P. syringae pv. tomato DC3000(pDCalgDP). As the HR progressed, histochemical staining indicated that individual bacterial cells on the surface of resistant tomato leaves were expressing algD. These results indicate that algD is expressed in both susceptible (e.g. collard, tomato) and resistant (Rio Grande-PtoR) host plants. The expression of algD in an incompatible host-pathogen interaction was further explored by monitoring transcriptional activity in leaves of tobacco, which is not a host for P. syringae pv. tomato. In tobacco inoculated with DC3000(pDCalgDP), an HR was evident within 12 h p.i., and algD expression was evident within 8-12 h p.i. However, when tobacco was inoculated with an hrcC mutant of DC3000, the HR did not occur and algD expression was substantially lower. These results suggest that signals that precede the HR may stimulate alginate gene expression in P. syringae. Histochemical staining with nitro blue tetrazolium indicated that the superoxide anion () is a signal for algD activation in planta. This study indicates that algD is expressed when P. syringae attempts to colonize both susceptible and resistant plant hosts.