Development of an agar-plug cultivation system for bioactivity assays of actinomycete strain collections

Natural products are an important source of lead compounds for the development of drug substances. Actinomycetes have been valuable especially for the discovery of antibiotics. Increasing occurrence of antibiotic resistance among bacterial pathogens has revived the interest in actinomycete natural product research. Actinobacteria produce a different set of natural products when cultivated on solid growth media compared with submersed culture. Bioactivity assays involving solid media (e.g. agar-plug assays) require manual manipulation of the strains and agar plugs. This is less convenient for the screening of larger strain collections of several hundred or thousand strains. Thus, the aim of this study was to develop a 96-well microplate-based system suitable for the screening of actinomycete strain collections in agar-plug assays. We developed a medium-throughput cultivation and agar-plug assay workflow that allows the convenient inoculation of solid agar plugs with actinomycete spore suspensions from a strain collection, and the transfer of the agar plugs to petri dishes to conduct agar-plug bioactivity assays. The development steps as well as the challenges that were overcome during the development (e.g. system sterility, handling of the agar plugs) are described. We present the results from one exemplary screening campaign targeted to identify compounds inhibiting Agr-based quorum sensing where the workflow was used successfully. We present a novel and convenient workflow to combine agar diffusion assays with microtiter-plate-based cultivation systems in which strains can grow on a solid surface. This workflow facilitates and speeds up the initial medium throughput screening of natural product-producing actinomycete strain collections against monitor strains in agar-plug assays.

Genome-wide association mapping and genomic prediction for adult stage sclerotinia stem rot resistance in Brassica napus (L) under field environments

Sclerotinia stem rot (SSR) is a fungal disease of rapeseed/canola that causes significant seed yield losses and reduces its oil content and quality. In the present study, the reaction of 187 diverse canola genotypes to SSR was characterized at full flowering stage using the agar plug to stem inoculation method in four environments. Genome-wide association study (GWAS) using three different algorithms identified 133 significant SNPs corresponding with 123 loci for disease traits like stem lesion length (LL), lesion width (LW), and plant mortality at 14 (PM_14D) and 21 (PM_21D) days. The explained phenotypic variation of these SNPs ranged from 3.6 to 12.1%. Nineteen significant SNPs were detected in two or more environments, disease traits with at least two GWAS algorithms. The strong correlations observed between LL and other three disease traits evaluated, suggest they could be used as proxies for SSR resistance phenotyping. Sixty-nine candidate genes associated with disease resistance mechanisms were identified. Genomic prediction (GP) analysis with all the four traits employing genome-wide markers resulted in 0.41–0.64 predictive ability depending on the model specifications. The highest predictive ability for PM_21D with three models was about 0.64. From our study, the identified resistant genotypes and stable significant SNP markers will serve as a valuable resource for future SSR resistance breeding. Our study also suggests that genomic selection holds promise for accelerating canola breeding progress by enabling breeders to select SSR resistance genotypes at the early stage by reducing the need to phenotype large numbers of genotypes.

Fungi from Galleries of the Emerald Ash Borer Produce Cankers in Ash Trees

The emerald ash borer (EAB, Agrilus planipennis) is a devastating invasive pest that has killed millions of ash trees in the United States and Canada. EAB was discovered in the US in 2002 and first reported in Minnesota in 2009. It attacks ash trees that are native to the United States, including Fraxinus americana (white ash), F. nigra (black ash) and F. pennsylvanica (green ash). It also attacks Chionanthus virginicus (white fringe tree). Seven species of fungi isolated and identified only from EAB-infested trees in a previous study as having the potential to cause cankers were used to test their pathogenicity in F. americana (white ash). The fungi used were Cytospora pruinosa, Diplodia mutila, Diplodia seriata, Paraconiothyrium brasiliense, Phaeoacremonium minimum, Phaeoacremonium scolyti, and Thyronectria aurigera. Two field experiments that used F. americana used two inoculation methods: woodchip and agar plug inoculations. Results indicated that all of the fungi tested caused cankers in varying amounts, as compared to the controls. The largest cankers were caused by D. mutila (270 mm2), C. pruinosa (169 mm2), and D. seriata (69 mm2). All fungi except for T. aurigera were re-isolated and sequenced to confirm Kochs’ postulates. Canker-causing fungi found in association with EAB galleries have the potential to contribute to tree dieback and mortality.

First report of Phytophthora nicotianae causing stem canker of Catalpa bungei (Chinese catalpa) in China

Chinese catalpa, Catalpa bungei C.A. Mey is native to China and has been widely cultivated as an important tree species for timber and ornamental purposes (Tao et al. 2019). The properties and high durability of the wood can resist the damage caused by microorganisms and insects (Xiao Y et al. 2019). In September 2020, stem cankers were observed in 5-year-old and 3-year-old C. bungei in a pilot experiment field covering 16-hectare area in Shuyang city (Jiangsu province, China) and in a nursery in Binhai city (Jiangsu Province, China), respectively. The disease incidence in both locations was about 1% to 3%. The typical disease symptoms include small to large, dark-brown and irregular-sunken canker around and along the stem under 2 meters from the stem base. The phloem and xylem of the symptomatic stem were dark brown and the xylem had larger necrosis than the phloem. The cross section of the diseased stem was partially died. The symptomatic stem were collected in both locations for pathogen isolation. In total, seven purified isolates from the diseased samples were obtained using potato dextrose agar (PDA) following standard isolation protocol (Huang et al. 2019). In order to determine the pathogenicity, 3-year-old Chinese catalpa seedlings were artificially inoculated with each of the seven isolates in April 2021. After removing the bark of the stem by a sterilized punch (diameter 6mm), an agar plug (diameter 6mm) pre-colonized by the isolate was inoculated to the stem and the inoculation point was sealed with parafilm. The agar plug without pre-colonization was used as control. Six tree seedlings were inoculated for each isolate. Ten days after inoculation, only the treatment with isolate QS.1 showed obvious discoloration around the inoculation point. One month after inoculation, the canker around the inoculation point was formed (3.4 cm ± 1.0 cm) and spread to the xylem, similar to the symptoms observed in the field. Isolate QS.1 was re-isolated successfully from the inoculated stem based on morphological characters, confirming the Koch's postulates and QS.1 as the causal pathogen. The isolate QS.1 formed white colonies with abundant aerial mycelia on V8 juice agar and produced a large amount of persistent and papillary ovoid sporangia with size of 22 ~ 45μm (average 31μm) × 18 ~ 39μm (average 23μm) in 10% aqueous solution of V8. The spore was spherical with thick-wall and diameter of 24 ± 3.9μm. The morphology of QS.1 is similar to that of Phytophthora nicotianae. The genomic DNA of representative isolate QS.1 was extracted from mycelium by a modified CTAB method (Murray et al. 1980). The rDNA internal transcribed spacer (ITS) region, β-tubulin and EF1-α genes were amplified and sequenced with primers ITS1/ITS4 (White et al. 1990), BTub_F1/TUBUR1 (L. et al. 2004) and EF1A_for/EF1A_rev (Blair et al. 2008), respectively. The BLAST results of these sequences (Accession No. MZ646302, MZ672116, and MZ675589, respectively) showed 99%, 100% and 100% identity with sequences of P. nicotianae (Accession No. KJ494902, KY205750, and MH359041), respectively. Based on the morphological characteristics and DNA analysis, isolate QS.1 was identified as P. nicotianae. To our best knowledge, this is the first report of P. nicotianae causing stem canker on Chinese Catalpa. This disease may pose potential threat on Catalpa due to the increase in Catalpa planting for economic and ecological purposes in China.

Phytophthora cactorum causing bleeding canker of Acer x freemanii in southern Idaho

Since 2018, bleeding cankers have been observed on maple trees in multiple home gardens in southwest Idaho. The cankers ooze a dark sap and and are approximately 10 cm to 35 cm in diameter. Cankers typically occur on the main trunk but are also present on scaffold branches in severe infecrions. Symptoms of foliar chlorois, branch dieback, and premature autumn senescence were also associated with the disease. Phytophthora DNA was detected in symptomatic material from five trees using real-time PCR (Miles et al., 2017). In July 2019 recovery of a causal agent from a symptomatic Acer x freemanii tree was attempted. Excisions were made from the interface of healthy and diseased tissue around the cankers using a chisel. The tissue was then placed in sealed plastic ziplock bags at 4°C for 7 days. Hyphae were then removed with forceps and placed onto potato dextrose agar (PDA) amended with penicillin G (0.2 g/liter) and streptomycin sulfate (0.8 g/liter). Colonies resembling Phytophthora cactorum were consistently observed after 5 days at 21°C. Tentative P. cactorum identification was based on the presence of abundant papillate and caducous sporangia on a short pedicel; sporangia were approximately 30 μm long and 26 μm wide (Bush et al., 2006; Hudler, 2013). Individual hyphal tips were transferred to fresh PDA plates and sequencing of both the rDNA ITS region and Cytochrome c oxidase subunit I (COI) was completed for a representative isolate (D19-130). DNA extraction, PCR and sequencing were as previously described (Woodhall et al. 2013; Robideau et al., 2011). The resulting DNA sequences for rDNA ITS (MW315449) and COI (MW881040) were both 100% identical (723/723 bp and 728/728 bp) with sequences from cultures previously identified as P. cactorum (MH171627 and MH136858). To determine pathogenicity, 14 month-old maple (A. x freemanii) trees in individual containers with potting mix were wounded 15 mm above the soil line with a single 10 mm incision using a sterile razor blade and inoculated by placing a 10 mm2 fully colonized PDA plug of isolate D19-130 on the wound. The inoculum and wound were then covered with a damp cotton ball that was secured loosely with parafilm. Control plants consisted of uninoculated plants and wounded plants inoculated with a PDA agar plug. Each treatment was replicated five times and placed in a controlled environment chamber set at 24ºC and 90% relative humidity. All treatments were sprayed with water daily to ensure the cotton balls remained damp. After 8 weeks, black lesions, up to approximately 25 mm above the soil line, were observed on the stem base of all P. cactorum-inoculated plants. No black lesions were observed on non-inoculated plants or plants inoculated with a PDA agar plug. P. cactorum was isolated from lesions, as described above, except polystyrene foam boxes containing moist paper towels were used instead of bags. This report confirms P. cactorum as a causal agent of bleeding canker of maple in Idaho for the first time. It has been shown that several Phytophthora species can infect maple (Jung and Burgess, 2009; Huddler, 2013). P. cactorum has a wide host range but certain strains have been associated with lethal bleeding stem cankers in maple and other deciduous trees worldwide (Huddler, 2013). Knowledge of the causal agent of bleeding canker on maple will help determine appropriate disease management practices.

Antimicrobial activity of endophytic fungi isolated from different plant parts of Curcuma mangga Valeton and Zijp

The endophytic fungi isolated from different plant parts including leaf, stem and rhizome of Curcuma mangga were screened for antimicrobial activity by employing agar plug diffusion assay and disc diffusion assay for primary screening and secondary screening, respectively. A total of 127 endophytic fungi that were successfully isolated from various plant parts were cultured to examine their antimicrobial activities. Qualitative screening using agar plug diffusion assay revealed that 118 isolates (92.9%) showed antimicrobial activity against at least on one test microorganisms and suggested that the rhizome part exhibited the highest percentage of antiyeast (58.3%) and antifungal (91.7%) activities compared to leaf and stem parts. Quantitative screening using disc diffusion assay indicated that ethyl acetate extract from fermentative broth (extracellular compound) demonstrated better antimicrobial activity compared to methanol extract derived from fungal biomass (intracellular compound) against all the four classes of pathogenic microorganisms tested (Gram-positive bacteria, Gram-negative bacteria, yeast and fungi). The future of endophytic fungus study is very promising as it possesses hidden potential to be developed as natural antimicrobial agent.

ANTIBACTERIAL ACTIVITY Streptomyces spp. ENDHOPHYTIC TO Staphylococcus aureus AND Eschericia coli

The objective of this research was to known antibacterial activity of 4 endhophytic Streptomyces namely Streptomyces griseorubiginosus, Streptomyces vellosus, Streptomyces diastaticus and Streptomyces griseoruber against Staphylococcus aureus and Eschericia coli and measure the biggest antibacterial activity which is indicate by clear zone diameter. Antibacterial activity by using agar plug diffusion method showed that Streptomyces vellosus has capable to inhibits growth of Staphylococcus aureus but not with Eschericia coli and Streptomyces diastaticus has capable to inhibits growth of Eschericia coli but not with Staphylococcus aureus. The clear zone of Streptomyces vellosus and Streptomyces diastaticus are 5,18 mm and 7 mm respectively. Streptomyces griseoruber and Streptomyces griseorubiginosus can’t grow during isolation. Keyword: Streptomyces vellosus, Streptomyces diastaticus, agar plug diffusion method

Bioprospecting of insect nest associated actinobacteria with special reference to antimicrobial and anti HIV activity

The present study attempted to explore actinobacteria from different insect nest samples for antimicrobial activity. Totally, 43 actinobacterial colonies were recovered from ant nest, termite nest, wasp nest and blanket worm nest samples by adopting standard spread plate method. Screening of antimicrobial properties of actinobacterial strains was determined by agar plug method. Two actinobacterial strains AN1 and AN5 showed promising activity (14-18 mm inhibition) against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, carbapenem resistant Klebsiella pneumoniae and Mycobacterium smegmatis. Both the strains produced antimicrobial compound earlier on ISP2 agar when compared to ISP2 broth. Crude extracts from the strains AN1 and AN5 were produced by adopting agar surface fermentation and extracted using ethyl acetate. Based on the studied phenotypic characteristics, actinobacterial strains AN1 and AN5 isolated from ant nest were identified as Streptomyces sp. In addition to antimicrobial activity, extracts also showed anti-HIV activity. This study concluded that insect nest is a promising source for bioactive actinobacteria. Two potential Streptomyces sp. AN1 and AN5 isolated from ant nest will be promising sources for antimicrobial metabolites against drug resistant bacteria, retrovirus and mycobacterial pathogens.

Genome-Wide Association Mapping and Genomic Prediction for Adult Stage Sclerotinia Stem Rot Resistance in Brassica Napus (L) Under Field Environments

Sclerotinia stem rot (SSR) is a fungal disease of rapeseed/canola that causes significant seed yield losses and reduces its oil content and quality. In the present study, the reaction of 187 diverse canola genotypes to SSR was characterized at full flowering stage using the agar plug to stem inoculation method in four environments. Genome-wide association study (GWAS) using three different algorithms identified 133 significant SNPs corresponding with 123 loci for disease traits like stem lesion length (LL), lesion width (LW), and plant mortality at 14 (PM_14D) and 21 (PM_21D) days. The explained phenotypic variation of these SNPs ranged from 3.6–12.1%. Nineteen significant SNPs were detected in two or more environments, disease traits with at least two GWAS algorithms. The strong correlations observed between LL and other three disease traits evaluated, suggest they could be used as proxies for SSR resistance phenotyping. Sixty-nine candidate genes associated with disease resistance mechanisms were identified. Genomic prediction (GP) analysis with all the four traits employing genome-wide markers resulted in 0.43–0.63 prediction accuracy. However, the prediction efficiency was further improved 0.55–0.88 when we integrated the GWAS information in the GP model. From our study, the identified resistant genotypes and stable significant SNP markers will serve as a valuable resource for future SSR resistance breeding. Our study also suggests that genomic selection holds promise for accelerating canola breeding progress by enabling breeders to select SSR resistance genotypes at the early stage by reducing the need to phenotype large numbers of genotypes.