Laboratory studies assessing the microbial biocontrol potential of diverse strains of Beauveria bassiana isolated from coffee berry borer, with emphasis on strains from Hawai'i Island and comparisons to commercial strain GHA.

A series of experiments investigated genetically diverse strains of Beauveria bassiana (Bb) isolated from coffee berry borer (CBB). Objectives included assessment of their biocontrol potential, particularly in comparison to Bb commercial strain GHA currently applied for CBB control, and identification of various attributes potentially contributing to their comparatively greater epizootic potential in CBB populations. Bioassays identified one strain from Hawai'i Island and one from Puerto Rico with virulence greater than GHA based on equal weights of unformulated conidial powder (CP); however, the greater potency of the CPs was ultimately explained by their 2.4-fold greater conidial densities (ca. 3.1 vs 1.3 × 1011 viable conidia/g CP). Density was explained, in large part, by conidial size, but not by size alone. Bb-inoculated CBB held on moist filter paper were more susceptible to infection than those held on cooked green coffee bean (CGCB). A Bb strain representative of the most common Hawaiian haplotype produced 2.6x more conidia after death of CGCB-held beetles than GHA (19.1 vs 7.3 x106 conidia/cadaver). Following host death, no difference was observed in time to emergence and initial conidial production by GHA and a selected group of Hawaiian strains; however, mass sporulation was initiated within 2 days by strain GHA compared to 4-5 days by the Hawaiian strains. In a preliminary evaluation of conidial mass-production potential, CP yields of several strains were comparable to GHA on a weight basis and significantly greater than GHA on a conidial basis (1.3-1.6 vs 0.7 × 1013 viable conidia/kg barley substrate).

New Ceratocystis species associated with rapid death of Metrosideros polymorpha in Hawai'i.

The native 'ohi'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 'ohi'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 'ohi'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.

Collection, isolation, in vitro culture, and laboratory transmission of Hirsutella eleutheratorum (Hypocreales: Ophiocordycipitaceae) from coffee berry borer on Hawai'i Island.

The insect pathogenic fungus Hirsutella eleutheratorum was first reported as a pathogen of coffee berry borer (CBB) Hypothenemus hampei in Colombia in 1993. A similar CBB pathogen identified as Hirsutella sp. was reported also from Colombia in 2007; attempts at isolation and in vitro culture of this fungus were unsuccessful. During 2016 and 2017 on the island of Hawai'i, extensive sampling of CBB populations was conducted in coffee fields treated with Beauveria bassiana-based biopesticides and in untreated fields. Among the samples collected from two high-elevation sites in the district of South Kona were rare findings of adult foundress CBB infected with a species of Hirsutella fitting the description of H. eleutheratorum. Prevalence of the pathogen was, in all cases, very low (<1%), having no significant impact on pest populations, even under conditions supporting epizootics of B. bassiana. The fungus was readily isolated from freshly-killed CBB and cultured on potato dextrose agar (PDA). Molecular characterization identified the fungus as a member of the Hirsutella citriformis clade, which includes species recently placed in the genus Ophiocordyceps. Adult CBB exposed to fungus-killed beetles or to PDA cultures of the fungus succumbed to infection within 10-14 days. Under high-humidity laboratory conditions, the fungus emerged from the killed host and produced long, conidia-bearing synnemata characteristic of the species. To our knowledge, this is the first record of H. eleutheratorum from CBB in Hawai'i and the first account of isolation, in vitro culture, genetic characterization, host-to-host transfer, and culture-to-host transfer of this fungal pathogen.

Prevalence of naturally-occurring strains of Beauveria bassiana in populations of coffee berry borer Hypothenemus hampei on Hawai'i Island, with observations on coffee plant-H. hampei-B. bassiana interactions.

Beauveria bassiana (Bb) strain GHA is a major component of an areawide pest management program for coffee berry borer (CBB) in Hawai'i. Recent studies have aimed to provide comprehensive assessments of the efficacy of the Bb-spray component of these programs for economic analyses; however, evaluations have been complicated by activity of naturally-occurring strains of this pathogen infecting CBB. Investigations were therefore undertaken to characterize these strains, assess their natural epizootic potential, and account for their contribution to CBB population suppression. A number of field sites were encountered with no history of significant use of commercial Bb-based biopesticides and where strain GHA was not detectable. Sampling of these sites was conducted early in the coffee season. Greatest activity of wild-type Bb strains was observed on high-elevation farms (>500 m), where 24-42% of foundress beetles in green coffee berries were infected. In contrast, infection rates did not exceed 4% on farms at low elevations (<300 m). Rates of 23-29% infection, comparable to those on high-elevation farms, were recorded in a stand of feral coffee at 293 m elevation, but the coffee was completely shaded and ventilation restricted by a dense overstory of vegetation. Despite high activity of naturally-occurring Bb at some sites (primarily sites at high elevations with humid, moderate-temperature environments and dense pest populations), these fungi did not prevent CBB from exceeding the economic threshold for commercial spray applications. Nevertheless, the high natural epizootic potential of these fungal strains suggests strong potential for development as microbial biocontrol agents.

First Report of Calonectria Leaf Spot on Ohelo in Hawaii.

Ohelo, Vaccinium reticulatum (Smith), is an endemic Hawaiian shrub, less than 1 m tall, which grows between 640 and 3,700 m elevation on disturbed volcanic sites on the islands of Maui and Hawaii (3). Ohelo berries are made into jams, jellies, and pie filling and are also a food source for the endemic nene goose, the state bird of Hawaii (3). In the summer of 2010, Ohelo berry plants grown in a greenhouse nursery located in Hilo, Hawaii, exhibited severe disease symptoms including leaf spots, stem lesions, and defoliation. The leaf spots appeared rapidly and were fairly severe. Subsequent field surveys of areas of naturally growing Ohelo within Hawaii Volcanoes National Park and along the roadside of Saddle Road were negative. An anamorphic state of Cylindrocladium was consistently isolated from the diseased portions of plants on potato dextrose agar (PDA). To determine the species, single-conidial isolates of the fungus were cultured for 14 days at 25°C under 12 h of light/dark conditions. Conidia were produced on penicillately branched condiophores having a stipe extension of 101.5 to 231.3 × 1.9 to 3.5 µm, terminating in a narrowly clavate vesicle, 2.4 to 3.5 µm. Conidia were hyaline, cylindrical, rounded at both ends, straight, three septate, and 58.6 to 77.77 × 4.29 to 5.72 µm. The nucleotide sequence of the partial ß-tubulin gene was determined for strain Vr1 and a BLAST analysis of the ß-tubulin sequence (GenBank Accession No. JX852715) revealed 99% similarity (337/341 bp) with the sequence of Calonectria pseudocolhounii strain CMW27213 (HQ285789) (1). Based on morphology and molecular sequencing, the fungus was identified as Calonectria irrespective of the teleomorphic stage in accordance with Lombard et al. (2). Koch's postulates were fulfilled by spray inoculating eight Ohelo seedlings and eight Ohelo variety N06-7 ('Kilauea') plants with a spore suspension (105 conidia per ml) of one isolate of the pathogen obtained from 14-day-old single-spore colonies grown on PDA at 25°C. Following inoculation, all plants were maintained in plastic bags in a growth chamber at 25 ± 1°C and 90 to 95% relative humidity. Four plants were used as a control. After 5 to 7 days, foliar symptoms resembling those seen in the nursery were detected on inoculated plants; leaf drop was first observed after day 7. No symptoms were detected on the control plants. The Calonectria sp. was reisolated from the artificially infected tissues. To our knowledge, this is the first report of a Calonectria sp. causing disease on Ohelo berry in Hawaii. References: (1) S. F. Chen et al. Persoonia 26:1, 2011. (2) L. Lombard et al. Stud. Mycol. 66:1, 2010. (3) F. Zee et al. F&N-16, May 2011.

First Report of Dolabra nepheliae Associated with Corky Bark Disease of Langsat in Hawaii.

In January 2011, branch samples were collected from langsat (Lansium domesticum Corr.), a fruit from Southeast Asia with an expanding niche market in Hawaii, exhibiting corky bark symptoms similar to that found on rambutan (Nephelium lappaceum) and litchi (Litchi chinensis) (3). The orchard, located along the Hamakua Coast of Hawaii Island, had 5- to 10-year-old trees, all with corky bark symptoms. As the trees matured, the cankers increased in size and covered the branches and racemes, often resulting in little to no fruit production. Scattered along the infected bark tissue were elongated, black ascomata present in the cracks. Ascomata were removed from the cracks using a scalpel blade, placed at the edge of a water agar petri dish and gently rolled along the agar surface to remove bark tissue and other debris. Individual ascomata were placed in 10-µl drops of 10% sodium hypochlorite on fresh water agar for 20 s, removed, and placed on potato dextrose agar petri dishes amended with 25 µg/ml streptomycin. The isolates were kept at 24°C under continuous fluorescent lighting. After 9 days, black pycnidia were present, which produced smooth, hyaline, linear to curved, filiform conidia, 4 to 6 septate (mostly 6), 31.8 to 70.1 × 2.0 to 2.8 µm. The morphological descriptions and measurements were similar to those reported for Dolabra nepheliae (3). The nucleotide sequence of the internal transcribed spacer (ITS) region including ITS1, 5.8S, and ITS2 intergenic spacers was determined for strain P11-1-1and a BLAST analysis of the sequence (GenBank Accession No. JX566449) revealed 99% similarity (586/587 bp) with the sequence of D. nepheliae strain BPI 882442 on N. lappaceum from Honduras. Based on morphology and ITS sequencing, the fungus associated with the cankers was identified as the same causal agent reported on rambutan and pulasan (N. mutabile) from Malaysia (1), and later reported on rambutan and litchi in Hawaii and Puerto Rico (3). Upon closer observations of the diseased samples, sections of corky bark contained at least two larval insects. The beetles were identified as Corticeus sp. (Coleoptera: Tenebrionidae) and Araecerus sp. (Coleoptera: Anthribidae) by the USDA-ARS Systematic Entomology Laboratory (Beltsville, MD). A corky bark disease on the trunk and larger limbs of mature langsat trees in Florida was thought to be caused by Cephalosporium sp. with larvae (Lepidoptera: Tineidae) feeding on the diseased tissue (2). It is not known the extent to which either of the beetle species is associated with L. domesticum in Hawaii or if they play a role in the bark disorder. To our knowledge, this is the first report of Dolabra nepheliae being found on langsat in Hawaii. Effective management practices should be established to avoid potential production losses or spreading the disease to alternative hosts. References: (1) C. Booth and W. P. Ting. Trans. Brit. Mycol. Soc. 47:235, 1964. (2) J. Morton. Langsat. In: Fruits of Warm Climates, p. 201-203. Julia F. Morton, Miami, FL, 1987. (3) A. Y. Rossman et al. Plant Dis. 91:1685, 2007.

First Report of Pestalotiopsis Leaf Blotch on Mangosteen in Hawaii.

Mangosteen (Garcinia mangostana L.) is a tropical evergreen tree that produces one of the most prized tropical fruits, commonly known as the "Queen of the Fruits.″ Mangosteen has the potential to occupy a rapidly expanding niche market in Hawaii. In October 2009, a disease was observed that produced brown leaf spots and blotches surrounded by bright yellow halos at a mangosteen orchard located in Hakalau, Hawaii (19° 53' 49″ N, 155° 7' 35″ W). Recently transplanted 10+ year old trees were 95 to 100% infected. Pieces of infected leaves and stems were surface-sterilized, plated on potato dextrose agar (PDA), and incubated at 24°C ± 1°C for 21 days. The fungus growing on PDA was pale buff with sparse aerial mycelium and acervuli containing black, slimy spore masses. Single spore isolates were used for the morphological characteristics and molecular analysis. Conidia were 5-celled. Apical and basal cells were hyaline; the three median cells were umber to olivaceous. Conidia (n = 50) were 24.3 ± 0.2 × 7.5 ± 0.1 µm, with apical appendages, typically three, averaging 24.3 ± 0.4 µm long, and a basal appendage averaging 6.7 ± 0.2 µm long. DNA sequences were obtained from the ß-tubulin gene and the internal transcribed spacer (ITS1 and ITS2) and 5.8S regions of the rDNA to confirm the identification. The morphological descriptions and measurements were similar to P. virgatula (Kleb.) Steyaert (1). Although sequence data of the ITS region (GenBank Accession No. JN542546) supports the identity of the fungus as P. virgatula, the taxonomy of this genus remains confused since there are only a few type cultures, so it is impossible to use sequences in GenBank to reliably clarify species names (2). To confirm pathogenicity, six leaves of two 3-year-old seedlings were inoculated. Seven-day-old cultures grown on 10% V8 agar at 24°C under continuous fluorescent lighting were used for inoculations. The inoculum consisted of spore suspensions in sterile distilled water adjusted to 6 × 105 conidia/ml. Using a fine haired paint brush, the inoculum was brushed onto the youngest leaves, while sterile distilled water was used as the control. The plants were incubated in a clear plastic bag placed on the laboratory bench at 24°C for 48 hours, then placed on a greenhouse bench and observed weekly for symptoms. After 14 days, leaf spots ranging in size from pinpoint to 5.4 mm in diameter with a distinctive yellow halo were present. Within 35 days, the leaf spots enlarged to leaf blotches ranging in size from 11.5 × 13.3 mm up to 28.3 × 34.6 mm with brown centers and a distinctive yellow halo identical to the field symptoms. A Pestalotiopsis sp. identical to that used to inoculate the seedlings was recovered from the leaf spots and blotches, confirming Koch's postulates. The experiment was repeated twice. Pestalotiopsis leaf blight has been reported in other countries growing mangosteen, including Thailand, Malaysia, and North Queensland, Australia (3). However, to our knowledge, this is the first report of a Pestalotiopsis sp. causing a disease on mangosteen in Hawaii. Although this disease is considered a minor problem in the literature (3), effective management practices should be established to avoid potential production losses. References: (1) E. F. Guba. Monograph of Pestalotia and Monochaetia. Harvard University Press, Cambridge, MA. 1961. (2) S. S. N. Maharachchikumbura et al. Fungal Div. 50:167, 2011. (3) R. C. Ploetz. Diseases of Tropical Fruit Crops. CABI Publishing. Wallingford, Oxfordshire, UK, 2003.

First Report of Pestalotiopsis virgatula Causing Pestalotiopsis Fruit Rot on Rambutan in Hawaii.

Rambutan (Nephelium lappaceum Linn.) is a tropical, exotic fruit that has a rapidly expanding niche market in Hawaii. Diseased rambutan fruit was commonly observed in orchards in the Hilo and Kona districts of Hawaii Island during 2006. In surveys conducted in January, symptoms appeared as dark brown-to-black spots on mature fruit and blackened areas at the base of spinterns (hair-like projections) of mature and immature fruits. Pieces of infected fruit (cv. R167) were surface sterilized for 2 min in 0.5% NaOCl, plated on potato dextrose agar, and incubated at 24 ± 1°C for 7 days. The fungus growing on PDA was pale buff with sparse, aerial mycelium and acervuli containing black, slimy spore masses. All isolates had five-celled conidia. Apical and basal cells were hyaline, while the three median cells were olivaceous; the upper two were slightly darker than the lower one. Conidia (n = 40) were 20.3 ± 0.1 × 6.8 ± 0.1 µm. There were typically three apical appendages averaging 16.8 ± 0.2 µm long. The average basal appendage was 3.8 ± 0.1 µm long. The fungus was initially identified as Pestalotiopsis virgatula (Kleb.) Stey. on the basis of conidial and cultural characteristics (3). The identification was confirmed by molecular analysis of the 5.8S subunit and flanking internal transcribed spacers (ITS1 and ITS2) of rDNA amplified from DNA extracted from single-spore cultures with the ITS1/ITS4 primers (1,4) and sequenced (GenBank Accession No. EU047943). To confirm pathogenicity, agar pieces, 3 mm in diameter, from 7-day old cultures were used as inoculum. Five mature fruit from rambutan cv. R134 were rinsed with tap water, surface sterilized with 0.5% NaOCl for 2 min, wounded with a needle head, inoculated in the laboratory, and maintained in a moist chamber for 7 days. Lesions resembling symptoms that occurred in the field were observed on fruit after 7 days. No symptoms were observed on fruit inoculated with agar media. The fungus reisolated from diseased fruit was identical to the original isolates, confirming Koch's postulates. The disease appears to be widespread in Hawaii. Preharvest symptoms may have the potential to affect postharvest fruit quality if fruits are not stored at the proper conditions. Pestalotiopsis spp. have been reported on rambutan in Malaysia, Brunei, and Australia (2). To my knowledge, this is the first report of P. virgatula causing fruit spots on rambutan in Hawaii. References: (1) G. Caetano-Annolles et al. Curr. Genet. 39:346, 2001. (2) D. F. Farr et al. Fungal Databases. Systematic Botany and Mycology Laboratory. On-line publication. ARS, USDA, 2007. (3) E. F. Guba. Monograph of Pestalotia and Monochaetia. Harvard University Press, Cambridge, MA, 1961. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. 1990.

Atypical Internal Yellowing of Papaya Fruit in Hawaii Caused by Enterobacter sakazakii.

Internal yellowing (IY) caused by Enterobacter cloacae and characterized by yellow discolored tissue surrounding the papaya (Carica papaya L.) seed cavity, diffuse margins, and the presence of a distinctly rotten odor was first reported in 1987 (3). Here we report the formation of atypical internal yellowing (AIY) in ripe papaya caused by the bacterium Enterobacter sakazakii. In surveys conducted from 2006 to 2007, 'Kapoho Solo' papayas grown in the Puna District of Hawaii Island were obtained from various packinghouses. After incubation at 27°C, the papayas were bisected and examined for symptoms of IY. Among papayas that were asymptomatic for IY, a dull, greenish yellow discoloration of the flesh with a distinct margin extending from the seed cavity into the pericarp was noted, along with a pungent odor. These symptoms occurred in 5 of the 500 fruit surveyed and bacterial populations were 102 to 103 CFU/g. Discolored tissue was aseptically excised, weighed, macerated, serially diluted in sterile distilled water (SDW), and plated onto modified peptone yeast extract medium (PT-M4) (4). The plates were incubated at 30°C for 24 to 48 h until single colonies were evident. After 48 h, colonies on PT-M4 were orange-red, convex and circular, and surrounded by a somewhat opaque 1-mm margin. After single colony purification, five strains were obtained. The strains, inoculated into oxidation/fermentation-glucose tubes and API 20E strips (bioMerieux, Inc., Durham, NC) incubated at 30°C, were shown to be facultative anaerobes and identified as E. sakazakii with a 98.4% certainty. Colonies plated onto tryptic soy agar (TSA) and incubated for 72 h at 25°C produced yellow pigmentation, indicative of E. sakazakii. Amplification by PCR with E. sakazakii-specific primers (2) yielded a 929-bp fragment, which was absent with E. cloacae and Pseudomonas aeruginosa template DNA. To confirm pathogenicity, cell suspensions at 109 CFU/ml of putative E. sakazakii strains RK07-05, RK07-06, and RK07-07 and E. cloacae (3) were inoculated by injection (0.5 ml per site) into one-third-ripe 'Kapoho Solo' papayas (six fruit per strain, inoculated at duplicate sites) and incubated at 27°C for 4 days. Control sites were injected with 0.5 ml of SDW. Fruit inoculation experiments were repeated. E. cloacae-inoculated sites produced typical IY as previously described (3), while the sites inoculated with the three E. sakazakii strains produced greenish yellow tissue (26% mean incidence), symptomatic of AIY. Control sites did not produce IY or AIY. Koch's postulates were fulfilled, and the identification of reisolated bacterial strains was confirmed with API 20E, PCR, and pigment production on TSA. Although less prevalent (1% incidence) than the typical IY produced by E. cloacae (3), E. sakazakii has the potential to affect quality and food safety of fresh and processed papaya products. E. sakazakii has been implicated in a severe form of neonatal meningitis, sepsis, and necrotizing enterocolitis (1). Research into the transmission and infection of papaya of this cross-domain pathogen merits further study. References: (1) D. H. Adamson. Clin. Microbiol. Newsl. 3:19, 1981. (2) A. Lehner et al. BMC Microbiol. 4:43, 2004. (3) K. A. Nishijima et al. Plant Dis. 71:1029, 1987. (4) K. A. Nishijima et al. Plant Dis. 88:1318, 2004.

Association of Enterobacter cloacae with Rhizome Rot of Edible Ginger in Hawaii.

Edible ginger is a popular spice crop that is grown in Hawaii primarily for the fresh market, and as such, rhizome quality is of paramount importance. In our studies, a Gram-negative, facultative anaerobic, rod-shaped bacterium was consistently isolated from decayed as well as symptomless ginger rhizomes. The bacterium was identified as Enterobacter cloacae by biochemical assays and 16S rDNA sequence analysis. Rot symptoms, which usually occurred in the central cylinder of the rhizome, were characterized by yellowish-brown to brown discolored tissue and firm to spongy texture. In inoculation experiments, ginger strains of E. cloacae produced basal stem and root rot, with foliar chlorosis and necrosis in tissue-cultured ginger plantlets, and discolored and spongy tissue in mature ginger rhizome slices and whole segments. In other hosts, ginger strains of E. cloacae caused internal yellowing of ripe papaya fruit and internal rot of onion bulbs. All strains that caused symptoms in inoculated plants were reisolated and identified as E. cloacae. Our studies suggest that E. cloacae can exist as an endophyte of ginger rhizomes, and under conditions that are favorable for bacterial growth, or host susceptibility, including maturity of tissues, rhizome rot may occur. Rhizome quality may be impacted by the presence of E. cloacae under conditions such as high temperature, high relative humidity, and low oxygen atmosphere that may affect the development of decay, and such conditions should be avoided during post-harvest handling and storage. The association of E. cloacae with a rhizome rot of ginger is a new finding.

Genetic divergence in the algT-muc operon controlling alginate biosynthesis and response to environmental stress in Pseudomonas syringae.

The algT-muc gene cluster (rpoE operon) is important for alginate production and survival during environmental stress in Pseudomonas syringae. The algT-muc operon was cloned and sequenced from P. syringae to determine whether the organization of this gene cluster was conserved in this plant pathogen. Interestingly, analysis of the algT-muc region in P. syringae revealed a unique arrangement when compared to other bacteria and lacked a mucC homologue. The relative importance of the mucC gene in the algT (rpoE) operon of various bacterial species is discussed.

AlgT (sigma22) controls alginate production and tolerance to environmental stress in Pseudomonas syringae.

Pseudomonas aeruginosa and the phytopathogen P. syringae produce the exopolysaccharide alginate, which is a copolymer of D-mannuronic and L-guluronic acids. One of the key regulatory genes controlling alginate biosynthesis in P. aeruginosa is algT, which encodes the alternate sigma factor, sigma(22). In the present study, the algT gene product from P. syringae pv. syringae showed 90% amino acid identity with its P. aeruginosa counterpart, and sequence analysis of the region flanking algT in P. syringae revealed the presence of nadB, mucA, and mucB in an arrangement virtually identical to that of P. aeruginosa. An algT mutant of P. syringae was defective in alginate production but could be complemented with wild-type algT from P. syringae or P. aeruginosa when expressed in trans. The algT mutant also displayed increased sensitivity to heat, paraquat, and hydrogen peroxide (H(2)O(2)); the latter two compounds are known to generate reactive oxygen intermediates. Signals for activation of algT gene expression in P. syringae were investigated with an algT::uidA transcriptional fusion. Like that in P. aeruginosa, algT transcription in P. syringae was activated by heat shock. However, algT expression in P. syringae was also stimulated by osmotic stress and by exposure to paraquat, H(2)O(2), and copper sulfate. The latter two compounds are frequently encountered during colonization of plant tissue and may be unique signals for algT activation in P. syringae.

dnaK and the heat stress response of Pseudomonas syringae pv. glycinea.

The dnaK gene from Pseudomonas syringae pv. glycinea PG4180 was cloned and sequenced. The dnaK coding region was 1,917 bp and contained a putative sigma 32 heat shock promoter 86 bp upstream of the translational start site. grpE, another heat shock gene, was found immediately upstream of the putative dnaK promoter. The predicted amino acid sequence of dnaK showed relatedness to the ATPase and substrate binding domains commonly found in heat shock proteins, as well as the highly conserved signature sequence motifs belonging to the Hsp70 protein family. Furthermore, the PG4180 dnaK gene complemented an Escherichia coli dnaK mutant for growth at temperatures above 37 degrees C, indicating that a fully functional dnaK homologue had been cloned from P. syringae pv. glycinea. All attempts to eliminate dnaK function by insertion mutagenesis failed, possibly because DnaK performs essential functions in P. syringae pv. glycinea. Expression of dnaK in P. syringae pv. glycinea PG4180 was investigated by constructing dnaK::uidA transcriptional fusions; expression of dnaK increased markedly when cells were preincubated at 18 degrees C and then shifted to 35 degrees C. An anti-DnaK monoclonal antibody was used to detect DnaK; in P. syringae pv. glycinea race 4, DnaK levels followed cell density during a 6-h incubation at 26 degrees C. When cells were shifted from 26 degrees C to either 32 or 38 degrees C, DnaK levels increased transiently, and then decreased rapidly. Although the cells continued to grow when incubated at 32 degrees C, growth was not supported at 38 degrees C. Our results indicate that P. syringae pv. glycinea responds to heat shock by producing DnaK, but DnaK does not aid in acclimation to sustained elevated temperatures.

Isolation of pigmentation mutants of the green filamentous photosynthetic bacterium Chloroflexus aurantiacus.

Mutants deficient in the production of bacteriochlorophyll c (Bchl c) and one mutant lacking colored carotenoids were isolated from the filamentous gliding bacterium Chloroflexus aurantiacus. Mutagenesis was achieved by using UV radiation or N-methyl-N'-nitro-N-nitrosoguanidine. Several clones were isolated that were deficient in Bchl c synthesis. All reverted. One double mutant deficient both in Bchl c synthesis and in the synthesis of colored carotenoids under anaerobic conditions was isolated. Isolation of a revertant in Bchl c synthesis from this double mutant produced a mutant strain of Chloroflexus that grew photosynthetically under anaerobic conditions and lacked colored carotenoids. Analysis of pigment contents and growth rates of the mutants revealed a positive association between growth rate and content of Bchl c under light-limiting conditions.

Saphenous vein stripping and its complications.

Saphenous vein stripping is one of the most common general surgical operations. Complications are rare and seldom serious; they can be fur- ther minimized by careful patient selection and meticulous technique. Adherence to the basic principles described should help further reduce the complication rate and maximize therapeutic benefit.

Use of Doppler ultrasound in intestinal surgery.

Doppler ultrasonography was used intraoperatively in 117 patients undergoing intestinal anastomosis or enterostomy to determine the adequacy of blood supply at the margins of resection. Doppler findings were compared with clinical assessment of intestinal blood flow by the operating surgeon. In 92 per cent of cases, Doppler signals and clinical observation coincided. However, in five of six cases in which Doppler signals were absent at one margin, the surgeon resected additional intestine, selecting margins within 1 cm of the nearest arterial Doppler signal. All five patients had uneventful healing. In the one case in which the surgeon chose to rely onthe appearance of the bowel despite the absence of Doppler arterial signals, ischemic necrosis of the proximal segment and anastomotic disruption occurred. The technique of Doppler ultrasonography is readily learned, and the instrument is available in most hospitals. Intraoperative use of Doppler ultrasonography can help identify intestine lacking a blood supply adequate to assure viability before changes in the appearance of the bowel alert the surgeon to the problem.

Parietal cell autoantibodies and hypergastrinemia in achlorhydria and the Zollinger-Ellison syndrome.

Parietal cell autoantibody (PCA), basal gastrin, and calcium-stimulated gastrin were measured in twenty patients with achlorhydria, in eight patients with the Zollinger-Ellison syndrome, and in fifty control subjects. In twelve patients with achlorhydria with a spared antrum, PCA was positive and basal gastrin was elevated. In contrast, eight achlorhydric patients with antral gastritis had negative PCA and significantly lower basal gastrin levels. Patients with the Zollinger-Ellison syndrome did not demonstrate positive PCA despite elevated levels of basal gastrin, nor was PCA present in normal controls. This study suggests that certain achlorhydric states are caused by an autoimmune response, particularly if antral function is spared.

Villous adenomas of the duodenum.

A patient with multiple villous adenomas of the duodenum is described. Endoscopy plays an essential role in the management of these neoplasms. If no evidence of invasive malignancy is found on multiple endoscopic biopsies, wide local excision is the initial procedure of choice. Invasive malignancy found in either the endoscopic biopsy or the surgical specimen is indication for pancreaticoduodenectomy.

Determination of viability of ischemic intestine by Doppler ultrasound.

Doppler ultrasound was used to determine the viability of ischemic small intestine and to select the optimum point for resection of nonviable bowel. Twenty ischemic segments of small intestine were produced in dogs by ligating the vascular supply. The Doppler ultrasound probe then was used to determine the last point of arterial flow within the bowel wall. The dogs were reexplored after 24 hours. Histological examination of full-thickness biopsies showed the intestine to be normal in all 20 segments at the last audible Doppler signal, and in 19 of the 20 segments at 1 cm distal to the last signal. Progressive degrees of necrosis were observed at 2 and 3 cm distal to the last signal. Twenty-five segments of ischemic intestine were resected in baboons. All resections performed at the last Doppler signal or 1 cm distal to it were normal 1 month later. Of 15 resections performed at 2, 3, and 4 cm distal to the last signal, 10 showed evidence of stricture or anastomotic disruption. Doppler ultrasound is a reliable method for determining the viability of ischemic intestine and for selecting the optimum point for resection of nonviable bowel.