Diversity of Endophytic Actinomycetes Producing Indole-3- Acetic Acid and In vitro Evaluation of Plant Growth-promoting Activity on Brassica oleracea L.

The evaluation of endophytic actinomycetes diversity, growth-promoting strain effects on cauliflower seeds germination, and in vitro organogenesis are the objectives in this study. Moreover, 15 strains from 125 isolates were determined to produce indole-3- acetic acid (IAA), where majority was obtained from roots (66.67%), followed by from branches (26.67%) and leaves (6.67%). Specifically, Jatropha sp. is a plant species with the most endophytic actinomycetes content compared to others. In addition, all endophytic Streptomyces strains were screened based on IAA production ability in vitro on yeast extract–malt extract broth (YMG) broth medium. The results showed the tendency for one strain with code Streptomyces sp. KMR-1E to generate a maximum IAA isolate from Cinnamomun sp. plant. Furthermore, the molecular taxonomy and phylogenetic analysis were recreated from 16S rRNA gene sequences, which attributed the KMR- 1E to genus Streptomyces. Meanwhile, plant growth promotion was evaluated under in vitro condition. This exposed the individual ability to enhance the shoot and root length of cauliflowers. The untreated cultures with a strain free agar block was used as control.

First Report of Binucleate Rhizoctonia AG-L Causing Root and Stem Rot of Wishbone Flower (Torenia fournieri) in Taiwan

Wishbone flower (Torenia fournieri L.) is a common ornamental plant for flower bed in Taiwan. In August 2018, root and stem rot of wishbone flower occurred on the flower bed in the campus of National Chung Hsing University, Taichung city, with 100% incidence. Symptoms were dark brown discoloration of basal stems and brown necrosis of roots. Lesions from base of stems were excised into 5 mm long fragments, which were then surface sterilized in 1% sodium hydrochloride for 1 min, rinsed in sterile distilled water, dried on filter paper and thereafter placed onto 2% water agar. After 24 h, hyphae characteristic of Rhizoctonia (Sneh et al. 1991) appeared and dominated in every isolation. Hyphal tips were transferred to potato dextrose agar (PDA). After 5 days of incubation at 28°C, characteristic brown colonies of Rhizoctonia (Sneh et al. 1991) were observed. Hyphal width was 4.29±0.52 μm. No sclerotia were visibly present after 21 days of growth on PDA at 28°C. Hyphae were stained by 0.3% safranin-O and 1% KOH. There were two nuclei in each hyphal compartment, suggesting a binucleate Rhizoctonia fungus. ITS sequence has been used as the best tool to identify specific anastomosis group (AG) of Rhizoctonia as shown by Sharon et al. (2006, 2008). ITS sequence was amplified using the primers Bd1a and ITS4 (Goka et al. 2009; White et al. 1990). Blast search analysis of this acquired sequence (acc. no. LC498494) revealed the highest similarity (98.75 to 99.83%) with the reference sequences (acc. nos. AB286934, AB286933, and AB196653) of binucleate Rhizoctonia AG-L, namely Ceratobasidium sp. AG-L. Pathogenicity test was carried out using seedlings of 4-week-old wishbone flower each grown in a pot of 6.35 cm diameter. To prepare the inoculum, a PDA agar block (6 mm in diameter) excised from the growing front of 5-day-old colony was transferred into a flask with 200 ml of potato dextrose broth (PDB) incubated in a shaker at 26°C and 120 rpm for 6 days. The PDB broth was then blended into slurry. Ten pots each with a seedling were inoculated by pouring 50 ml of slurry onto the potting medium. Five pots were served as the controls by pouring PDB only. Pots were maintained in a greenhouse (26 to 33°C). Three days after inoculation, all inoculated plants exhibited symptom of root and stem rot. The same fungus was reisolated and confirmed by sequencing rDNA-ITS. This is the first report of root and stem rot of wishbone flower caused by binucleate Rhizoctonia AG-L in Taiwan and in the world. Although this is the second cases, since Wang and Hsieh (1993), for binucleate Rhizoctonia AG-L to be pathogenic, this study shows that this fungus has the potential to cause damages and is worth of further investigations.

ANTIFUNGAL ACTIVITY OF THE GENUS BACILLUSBACTERIA AS AGAINST THE PHYTOPATHOGEN ALTERNARIA SP.

Alternaria blights caused by microscopic fungi of the Al-ternariagenus are widespread diseases of crops and or-namental plants. Russian and foreign scientists take an active part in developing biological products based on an-tagonistically active microorganisms. However, the situa-tion regarding Alternaria blight is still unfavorable. The re-search goal was to study the antifungal activity of the ge-nus Bacillusstrains against the phytopathogen Alternaria sp. The agar block method was used to determine the an-tagonistic activity of 8 rhizospheric bacilli strains (B. pu-milus4, B. pumilus5, B. pumilus6, B. pumilus7, B. licheni-formis8, B. licheniformis9, B. licheniformis10, and B. pu-milus16). All investigated strains showed themselves as antagonists in relation to Alternaria. On the 14th day of the experiment, the diameter of the Alternaria sp. in the control was 85.83 ± 8.78 mm. The following values of the phyto-pathogen mycelium diameter were recorded in dishes with bacilli: B. pumilus4 -10.00 ± 0.87 mm, B. pumilus 5-12.17 ± 0.76 mm, B. pumilus 6-11.33 ± 1.26 mm, B. pu-milus7 -8.00 ± 3.00 mm, B. pumilus16 -7.67 ± 0.29 mm. Alternariasp. did not grow beyond the 5 mm diameter block with all B. licheniformisstrains. Bacteria of the B. licheniformisspecies had a more pronounced antifungal effect (100%) than the B. pumilusstrains (91.13-96.70%). B. licheniformis8, 9, 10 and B. pumilus16, 7, 4 strains are primarily recommended for inclusion in a biological plant protection product against Alternariablight

Screening of Soil Streptomycetes – Producers of Antibiotics against Phytopathogenic Bacteria

Soil is an inexhaustible source of bacteria of the genus Streptomyces – the producers of the vast majority of known antibiotics that are successfully used in medicine, veterinary and agriculture. The emergence and spread of pathogenic bacteria resistance to antibiotics requires the search for new antibiotic compounds capable of overcoming this problem. Aim. The purpose of this work was to isolate streptomycetes from soil samples of Kyiv and the Kyiv region and study their antibiotic activity against four strains of the different species of phytopathogenic bacteria. Methods. A suspension of soil in distilled water was sown on solid Chapek or corn-soybean medium in Petri dishes, in which trimethoprim and nystatin were introduced to inhibit bacterial and fungal growth. The antibiotic activity of the streptomycetes was tested by setting their agar discs on lawns of phytopathogenic bacteria in Petri dishes. Antibiotics were extracted from the streptomycetes agar cultures with a mixture of chloroform and acetone (2:1), dried in a rotary vacuum evaporator, dissolved in ethanol, separated and purified by thin layer chromatography on aluminum plates (Silica gel 60 F254 from Merck KGaA). The UV/Vis absorption spectra of the antibiotics were measured with a Beckman DU 8 spectrophotometer. Results. 10 strains of streptomycetes were isolated from the soil samples of Kyiv and the Kyiv region, whose antibiotic activity was tested against four phytopathogenic bacteria using the agar block method. Three of the streptomycetes – B8, SK and KZ, formed growth inhibition zones of different phytopathogens on complete medium in Petri dishes, among which the strain SK was the most active. This strain showed antibiotic activity against all four phytopathogenic bacteria – P. syringae 8511, P. carotovorum 8982, C. michiganensis 10 and X. campestris 8003. Conclusions. The results obtained are of interest for the protection of sensitive plants by isolated antibiotics against phytopathogenic bacteria in hothouse conditions.

Antibacterial and Antifungal Effects of Carica papaya and Cucurbita specie Seed Extracts on Escherichia coli and Aspergillus flavus

Objective: In this work, we studied the antifungal and anti-bacterial properties of seeds of Carica papaya and Curcubita specie using selected bacteria and fungi. Methods: Modified cold extraction method with ethanol and n-hexane was conducted. Antimicrobial properties of the extracts were done using agar block dilution for fungi and agar diffusion method for the bacteria. Measurement of the mean growth rate (MGR) for the fungi isolate and the inhibition zone diameter (IZD) for the bacteria were used as parameters. Results: Significant antifungal property was observed in ethanolic extract of Carica papaya at a concentration of 6% at four days of its exposure, while n-hexane extract of Carica papaya and ethanolic extract of Curcubita specie show fungistatic action. Ethanolic extract of Carica papaya at 6% concentration showed more antifungal property than the control drug. Antibacterial action for all the test extracts was poor, with the control drug showing more significant action than the extracts. There was a statistical significance difference between the ethanolic extract of Carica papaya and Curcubita specie (p< 0.05). Conclusion: This is an indication that ethanolic extract of Carica papaya can be used in the treatment of some of the fungal infection caused by Aspergillus flavus likewise n-hexane extracts. Keywords: Antifungal, Anti-bacterial, Carica papaya, Curcubita specie, ethanolic extract, n-hexane extract.

Antimicrobial activities and phylogenetic study of bacteria associated with Cyperus rotundus rhizosphere from Cemoro Sewu Plateau, Indonesia

Ambarwati A, Wahyuono S, Moeljopawiro S, Sembiring L, Yuwono T. 2019. Antimicrobial activities and phylogenetic study of bacteria associated with Cyperus rotundus rhizosphere from Cemoro Sewu Plateau, Indonesia. Biodiversitas 20: 2206-2212. A study has been conducted to investigate the activity of bacteria isolated from the rhizosphere of Purple Nut Sedge (Cyperus rotundus) from Cemoro Sewu Plateu, Indonesia as antimicrobial agent and the molecular relatedness between the isolates with other bacteria based on phylogenetic tree analysis. A total of six bacteria were obtained and tested for their capability to inhibit the growth of test organisms using agar block method and characterized molecularly using 16S rDNA sequence analysis. It was observed that the six isolates demonstrated the ability to inhibit at least one of four test microorganisms growth, with a diameter of inhibition zone ranging from 8-35 mm. The CRC32 isolate was the best isolate in inhibiting Staphylococcus aureus ATCC 25923 with a diameter inhibition zone of 26 mm (strong), while isolate CRA8 was the best anti-Candida (showing 35 mm of inhibition zone). The phylogenetic tree analysis resulted in two genera, four isolates belonging to Bacillus, while the other two isolates were known related to Paenibacillus genus. BLAST analysis on the CRC 32 isolate revealed that it is closely related with Paenibacillus peroriae strain 3763 with 99% sequence similarities and CRA8 isolates demonstrated the highest similarity with Paenibacillus sonchi strain X19-5 (97%). The results of this study demonstrated that the bacterial isolates from plant rhizosphere of Cemoro Sewu Plateau were the members of the Bacillus and Paenibacillus genera and demonstrated the potential as antimicrobial agent.

The Potential of Breadfruit Seed and Jackfruit Seed as Alternative Replacement Medium of Potato Dextrose Agar (PDA) with Seedling F0 Mushrooms

Potato Dextrose Agar (PDA) is the most media to grow the mushrooms, but the price of this media is expensive. Thus we need alternative media that easy and cheap to get it. This study aims to determine the growth of Aspergillus niger in alternative Artocarpus communis and Artocarpus heterophyllus seeds media. This research was an experimental study using a completely randomized design (CRD) one factor was the type of media is PDA (M0), Artocarpus communis seed media (M1), Artocarpus heterophyllus seed media (M2) and using the test mushrooms A. niger (J1). Inoculation of A. niger used agar block method for 3 days with a temperature of 28°C. Parameter of research was colony diameter and sporulation of A. niger. Data obtained with qualitative and quantitative methods. The result of this research showed that the best growth for Aspergillus niger was after 72 hours incubation. Colony diameter continually in PDA media, Artocarpus communis media, and Artocarpus heterophyllus media is 4.7 cm, 4.3 cm, and 4.1 cm with heavy sporulation. Therefore, Artocarpus communis and Artocarpus heterophyllus seeds media can be utilized as a substitution of PDA media for the growth of mushrooms.

Diagnosis Hama dan Penyakit Tanaman Bawang Merah Menggunakan Sistem Pakar

Penelitian ini mencoba membuat sebuah sistem pakar untuk mendeteksi hama dan penyakit yang ada pada tanaman bawang merah dengan merancang sebuah design secara maksimal dengan menggunakan DFD, ERD, serta block diagram untuk menemukan sistem yang bisa digunakan untuk mendiagnosa hama dan penyakit pada tanaman bawah merah, adapun teknik diagnosa yang digunakan dalam penelitian ini menggunakan forward chaining, penelusurannya dimulai dari fakta-fakta terlebih dahulu baru menjurus pada sebuah kesimpulan, Sedangkan Block diagram yang dibuat berfungsi untuk membatasi area permasalahan dalam penelitian ini, untuk menerapkan block diagram kedalam sebuah sistem database selanjutnya adalah merancang DFD dan ERD agar block diagram bisa diterapkan pada database dan aplikasi.

Quantitative visualization of gene expression inPseudomonas aeruginosaaggregates reveals peak expression of alginate in the hypoxic zone

It is well appreciated that oxygen- and nutrient-limiting gradients characterize microenvironments within chronic infections that foster bacterial tolerance to treatment and the immune response. However, determining how bacteria respond to these microenvironments has been limited by a lack of tools to study bacterial functions at the relevant spatial scalesin situ. Here we report the application of the hybridization chain reaction (HCR) v3.0 toPseudomonas aeruginosaaggregates as a step towards this end. As proof-of-principle, we visualize the expression of genes needed for the production of alginate (algD) and the dissimilatory nitrate reductase (narG). Using an inducible bacterial gene expression construct to calibrate the HCR signal, we were able to quantifyalgDandnarGgene expression across microenvironmental gradients both within single aggregates and within aggregate populations using the Agar Block Biofilm Assay (ABBA). For the ABBA population, alginate gene expression was restricted to hypoxic regions within the environment (~40-200 μM O2), as measured by an oxygen microelectrode. Within individual biofilm aggregates, cells proximal to the surface expressed alginate genes to a greater extent than interior cells. Lastly, mucoid biofilms consumed more oxygen than nonmucoid biofilms. These results establish that HCR has a sensitive dynamic range and can be used to resolve subtle differences in gene expression at spatial scales relevant to microbial assemblages. Because HCR v3.0 can be performed on diverse cell types, this methodological advance has the potential to enable quantitative studies of microbial gene expression in diverse contexts, including pathogen behavior in human chronic infections.ImportanceThe visualization of microbial activities in natural environments is an important goal for numerous studies in microbial ecology, be the environment a sediment, soil, or infected human tissue. Here we report the application of the hybridization chain reaction (HCR) v3.0 to measure microbial gene expressionin situat single-cell resolution in aggregate biofilms. UsingPseudomonas aeruginosawith a tunable gene expression system, we show that this methodology is quantitative. Leveraging HCR v3.0 to measure gene expression within aP. aeruginosaaggregate, we find that bacteria just below the aggregate surface are the primary cells expressing genes that protect the population against antibiotics and the immune system. This observation suggests that therapies targeting bacteria growing with small amounts of oxygen may be most effective against these hard-to-treat infections. More generally, HCR v3.0 has potential for broad application into microbial activitiesin situat small spatial scales.