First Report of Gnomoniopsis idaeicola Causing Cane Wilt and Canker in Commercial Blackberry Fields in Oregon

Oregon is the leading producer of blackberries in the United States (USDA 2021). In November 2020, we visited a seven-year-old, 5.6 HA field of thornless trailing cultivar ‘Columbia Star’ in Marion County, Oregon in response to grower reports of rapid plant death that they called ‘Blackberry Collapse.’ The pattern of disease in the field was semi-circular patches of dead plants. We estimate that ca. 17% of the plants were killed. Symptoms included fewer and stunted primocanes, floricane reddening, premature floricane senescence, or elliptical or irregular brown to purple cane lesions often near the base of necrotic petioles clasping the cane. Twenty-two cane lesions were collected, surface-disinfested, and placed on potato dextrose agar with streptomycin. In addition to common Rubus cane pathogens (Kalmusia coniothyrium, Botrytis spp., Diaporthe spp., and Seimatosporium lichenicola), Gnomoniopsis idaeicola was isolated from 23% of the samples. After isolation of G. idaeicola from ‘Columbia Star’, we also isolated the pathogen from symptomatic ‘Black Diamond’ blackberry in fields in Washington and Linn Counties in Oregon. G. idaeicola grew as white- to cream-colored circular colonies on the agar surface with sparse to dense aerial hyphae. Some isolates produced cream-colored exudate from irregular conidiomata. Conidia were single-celled, hyaline, oval to ellipsoid, and ranged from 4.9 to 6.9 µm long and 1.2 to 2.5 µm wide (n = 100). Perithecia and ascospores were not seen on canes or in culture; molecular methods were used to identify the fungus. Genomic DNA was extracted from nine isolates and β-tubulin, ITS region, and tef-1α were amplified using the primers and conditions described by Walker et al. (2010). Amplicons were Sanger sequenced in both directions by the Core Facilities of the Center for Quantitative Life Sciences of Oregon State University, Corvallis, OR. Sequences were deposited in GenBank (β-tubulin OK539758-OK539766; ITS OK348854-OK348862; tef-1α OK539767-OK539775). β-tubulin sequences (737 bp) were 100% identical to sequence of G. idaeicola from Rubus procerus, Oregon (GU320783). ITS sequences (518 bp) were 99.2 to 100% identical to G. idaeicola CBS125674T (GU320796). tef-1α sequences (860 bp) were 99.9 to 100% identical with reference sequences of G. idaeicola. Phylogenetic analyses of concatenated sequences using Tamura-Nei neighbor-joining (Tamura et al. 2004) confirmed identity. Pathogenicity of G. idaeicola isolates was tested in repeated experiments on four to five detached ‘Black Diamond’ primocanes and potted ‘Columbia Star’ plants with 4 mm hyphal plugs placed on fresh wounds and wrapped in parafilm; controls were treated with sterile agar plugs (Ellis et al. 1984; Stevanović et al. 2019). By 14 days, in each experiment, expanding necrotic lesions were observed on canes inoculated with G. idaeicola, but not with sterile agar plugs. ITS-sequence of fungi isolated from lesions confirmed identity as G. idaeicola, fulfilling Koch’s postulate. G. idaeicola was described as a new species by Walker et al. (2010) from perithecia collected from wild Rubus spp. in the Pacific Northwest. Despite its presence on native Rubus, G. idaeicola has not been reported in commercial blackberry fields in OR. Stevanović et al. (2019) identified G. idaeicola as an important pathogen causing canker, wilt, and death of blackberry in Serbia. Surveys of Oregon blackberry fields for G. idaeicola are ongoing, along with research on the epidemiology and management of this emerging pathogen.

The role of flagella in Pseudomonas-Pedobacter social motility across a surface

Bacteria often reside in multi-species communities where many behaviors result from interspecies relationships. In a two-species community, we show that co-culture of Pseudomonas fluorescens and Pedobacter sp. permits motility across a hard agar surface where neither species moves alone. Pseudomonas species engage in surface motility, including swimming and swarming, but these require moist environments. We are exploring the role of the Pseudomonas flagella in social motility. We deleted genes related to flagellar structure and function to study the importance of flagellar elements on the social phenotype. Using microscopy and swimming assays, we evaluate the effects of gene deletions on the presence and function of flagella in the resulting mutants, and evaluate the effect on social motility by observing the phenotype on both hard (2% w/v) and soft agar (1% w/v). Removal of the flagellar filament abolishes social motility, indicating a requirement for flagella in social motility. Removal of the flagellar motor also abolishes social motility, demonstrating that flagella must be functional. However, removal of membrane-spanning structural components, or part of the type III secretion system, results in mutants that lack flagella, but participate in a similar motile behavior with Pedobacter. Here we describe a role for flagella in motility of a two-species consortium across a hard agar surface, an environment considered non-permissive for flagellar motility. The requirement for both bacterial species indicates we are observing motility as a social phenotype, with a contribution from Pedobacter that enables the Pseudomonas flagella to function under conditions relevant in the natural soil environment.

KOSAKONIA SP. PROTEOLYTIC BACTERIA ISOLATED FROM RUMEN AND RETICULUM OF ACEH CATTLE

The aim of this study was to identify proteolytic bacteria from the ruminal and reticulum fluids of aceh cattle based on the 16S rRNA gene. Samples used were ruminal and reticulum fluids of aceh cattle slaughtered in Abattoir of Aceh Besar. Samples were diluted and cultured into Skim Milk Agar medium at 39 C for 48 hours. The morphology of bacterial colonies growth in the medium was observed. Colonies resulted in the largest clear zone were isolated and used for Deoxyribonucleic Acid (DNA) isolation, 16S rRNA gene amplification and sequencing. Theresults showed that morphology of dominant colonies was yellowish white color, round shape, position on the agar surface. The results ofphylogenetic analysis of RS1 and ReS2 isolates isolated from rumen and reticulum fluids of aceh cattle respectively had a close familialrelationship and belonged to the bacterial group of Kosakonia. Sequence homology showed isolate RS1 and ReS2 are probably either newEnterobacteriaceae species or unconfirmed species. Halo zone produced by ruminal bacteria had a wider diameter (25 mm vs 20 mm) than thatcaused by reticulum bacteria). Based on the results, RS1 (bacterium in the rumen) and ReS2 (bacterium in the reticulum) belong to similar type, namely Kosakonia sp. with a proteolytic activity. Presumably, these bacteria originate from the rumen that enters the reticulum with degraded feed.

Examination of a first-in-class bis-dialkylnorspermidine-terphenyl antibiotic in topical formulation against mono and polymicrobial biofilms

Biofilm-impaired tissue is a significant factor in chronic wounds such as diabetic foot ulcers. Most, if not all, anti-biotics in clinical use have been optimized against planktonic phenotypes. In this study, an in vitro assessment was performed to determine the potential efficacy of a unique first-in-class series of antibiofilm antibiotics and compare outcomes to current clinical standards of care. The agent, CZ-01179, was formulated into a hydrogel and tested against mature biofilms of a clinical isolate of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa ATCC 27853 using two separate methods. In the first method, biofilms were grown on cellulose discs on an agar surface. Topical agents were spread on gauze and placed over the biofilms for 24 h. Biofilms were quantified and imaged with confocal and scanning electron microscopy. In the second method, biofilms were grown on bioabsorbable collagen coupons in a modified CDC biofilm reactor. Coupons were immersed in treatment for 24 h. The first method was limited in its ability to assess efficacy. Efficacy profiles against biofilms grown on collagen were more definitive, with CZ-01179 gel eradicating well-established biofilms to a greater degree compared to clinical standards. In conclusion, CZ-01179 may be a promising topical agent that targets the biofilm phenotype. Pre-clinical work is currently being performed to determine the translatable potential of CZ-01179 gel.

Isolation, characterization and identification of antibiofouling metabolite from mangrove derived Streptomyces sampsonii PM33

In this study, we report the production, bioassay guided isolation and identification of antibiofouling metabolite from mangrove derived actinobacterium, Streptomyces sampsonii (PM33). The actinobacterial strain PM33 yields maximum amount of antifouling compounds through agar surface fermentation. In optimization, carbohydrates such as glucose, fructose and xylose, are suitable for maximum production of the active compound. In addition, other compounds such as malt extract, glutamine, and sodium chloride concentrations (2.5, 5 and 7.5%) and parameters such as pH 7.0 and temperature range 30 °C to 40 °C enhanced the production of antifouling metabolite. The antifouling metabolite was extracted in ethyl acetate. TLC and bioautography was used to separate and detect the antifouling metabolite present in the crude extract. The physico chemical features revealed that the antifouling metabolite PM33 – B as taxifolin (C15H12O7). The purified taxifolin was found to be active against biofouling bacteria, algal spore germination and mollusc foot adherence, respectively. Toxicity nature of taxifolin was also determined by adopting zebrafish embryos. The taxifolin isolated from mangrove-derived Streptomyces sampsonii PM33 is a promising candidate for the development of eco-friendly antifouling preparation.

Exploration of social spreading reveals behavior is prevalent among Pedobacter and P. fluorescens isolates, and shows variations in induction of phenotype

Within soil, bacteria are naturally found in multi-species communities, where interactions can lead to emergent community properties. It is critical that we study bacteria in a social context to investigate community-level functions. We previously showed that when co-cultured, Pseudomonas fluorescens Pf0-1 and Pedobacter sp. V48 engage in interspecies social spreading on a hard agar surface, a behavior which required close contact and was dependent on the nutritional environment. In this study, we investigate whether the ability to participate in social spreading is widespread among P. fluorescens and Pedobacter isolates, and whether the requirements for interaction vary. We find that this phenotype is not restricted to the interaction between P. fluorescens Pf0-1 and Pedobacter sp. V48, but is a more prevalent behavior found in one clade in the P. fluorescens group and two clades in the Pedobacter genus. We also discovered that the interaction with certain Pedobacter isolates occurred without close contact, indicating induction of spreading by a putative diffusible signal. As is the case for ISS by Pf0-1+V48, motility of all interacting pairs is influenced by the environment, with no spreading behaviors observed under high nutrient conditions. While Pf0-1+V48 require low nutrient but high NaCl conditions, in the broader range of interacting pairs this requirement for low nutrient and high salt was variable. The prevalence of motility phenotypes observed in this study and found within the literature indicates that community-induced locomotion in general, and social spreading in particular, is likely important within the environment. It is crucial that we continue to study microbial interactions and their emergent properties to gain a fuller understanding of the functions of microbial communities.

Interspecies interactions in bacterial colonies are determined by physiological traits and the environment

Interspecies interactions in bacterial biofilms have important impacts on the composition and function of communities in natural and engineered systems. To investigate these interactions, synthetic communities provide experimentally tractable systems. Agar-surface colonies are similar to biofilms and have been used for investigating the eco-evolutionary and biophysical forces that determine community composition and spatial distribution of bacteria. Prior work has focused on intraspecies interactions, using differently fluorescent tagged but identical or genetically modified strains of the same species. Here, we investigated how physiological differences determine the community composition and spatial distribution in synthetic communities of Pseudomonas aeruginosa, Pseudomonas protegens and Klebsiella pneumoniae. Using quantitative microscopic imaging, we found that interspecies interactions in multispecies colonies are influenced by type IV pilus mediated motility, extracellular matrix secretion, environmental parameters and the specific species involved. These results indicate that the patterns observable in mixed species colonies can be used to understand the mechanisms that drive interspecies interactions, which are dependent on the interplay between specific species’ physiology and environmental conditions.

Persistence of Candida auris on latex and nitrile gloves with transmission to sterile urinary catheters‡

Candida auris’ ability to persist on contaminated gloves and transmit to urinary catheters was evaluated. 105 and 103 cfu/ml suspensions of eight Candida species including C. auris were inoculated on latex and nitrile gloves fingertips and touched on agar surface at different time intervals. Urinary catheter piece, touched by latex glove carrying Candida spp. suspensions at various time intervals, was cultured by roll-plate method. C.auris persisted on latex gloves at both 105 and 103 cfu/ml up to 3 minutes and could be transmitted from both wet and dry contaminated gloves to catheters. Proper glove use with strict hand hygiene should be advocated in settings with ongoing C.auris transmission.

GROWTH RESPONSE OF Ganoderma sp. MYCELIUM TREATED WITH ROOT EXUDATES OF HERBACEOUS PLANTS

The purpose of the study was to study response of Ganoderma sp. that were given exudate treatment of plant roots. Ganoderma sp. is a pathogen that causes stem rot at base of oil palm. In addition to oil palm this fungus can attack hard and woody crops such as coconut, rubber, tea, cocoa etc. Isolate used is Ganoderma sp. Bio-10197 code obtained from Phytopathology laboratory SEAMEO BIOTROP Bogor. Mycelium Ganoderma sp. reproduced in malt agar media until mycelium grows over agar surface. Ganoderma mycelium was inoculated on 1x1x5 cm rubber wood pieces for 14 days until the mycelium grew over rubber sticks. The exudate used from ganyong (Canna edulis Kerr), garut (Maranta arundinacea Linn.), Ginger (Zingiber officinale Rosc.), Turmeric (Curcuma domestica Val.), Galangal (Alpinia galanga (L.) Sw.) and lidah mertua (Sansevieria trifasciata). The design used in this study was a complete randomized design (RAL) with 7 treatments consisting of 5 repeatations. The results of this study indicate that the mycelium experienced inhibition of growth, especially in the treatment of root exudate galangal shown by 60,81% percentage and tongue-in-law with a percentage of 59,58% colonization. On observation of growth characteristics of mycelium Ganoderma sp. visible zone delimited in the form of a brown mycelium pile suspected as an indication of mycelium rejection of bioactive compounds contained exudate.