Phenotypic and Genomic Comparison of Klebsiella pneumoniae Lytic Phages: vB_KpnM-VAC66 and vB_KpnM-VAC13

Klebsiella pneumoniae is a human pathogen that worsens the prognosis of many immunocompromised patients. Here, we annotated and compared the genomes of two lytic phages that infect clinical strains of K. pneumoniae (vB_KpnM-VAC13 and vB_KpnM-VAC66) and phenotypically characterized vB_KpnM-VAC66 (time of adsorption of 12 min, burst size of 31.49 ± 0.61 PFU/infected cell, and a host range of 20.8% of the tested strains). Transmission electronic microscopy showed that vB_KpnM-VAC66 belongs to the Myoviridae family. The genomic analysis of the phage vB_KpnM-VAC66 revealed that its genome encoded 289 proteins. When compared to the genome of vB_KpnM-VAC13, they showed a nucleotide similarity of 97.56%, with a 93% of query cover, and the phylogenetic study performed with other Tevenvirinae phages showed a close common ancestor. However, there were 21 coding sequences which differed. Interestingly, the main differences were that vB_KpnM-VAC66 encoded 10 more homing endonucleases than vB_KpnM-VAC13, and that the nucleotidic and amino-acid sequences of the L-shaped tail fiber protein were highly dissimilar, leading to different three-dimensional protein predictions. Both phages differed significantly in their host range. These viruses may be useful in the development of alternative therapies to antibiotics or as a co-therapy increasing its antimicrobial potential, especially when addressing multidrug resistant (MDR) pathogens.

First Report of Powdery Mildew Caused by Erysiphe cruciferarum on Camelina sativa in Montana

Camelina sativa (L.) Crantz, also known as false flax, is an annual flowering plant in the family Brassicaceae and originated in Europe and Asia. In recent years, it is cultivated as an important biofuel crop in Europe, Canada, and the northwest of the United States. In June of 2021, severe powdery mildew was observed on C. sativa ‘Suneson’ plants under greenhouse conditions (temperature 18.3°C/22.2°C, night/day) in Bozeman, Montana (45°40'N, 111°2'W). The disease incidence was 80.67% (150 pots, one plant per pot). White ectophytic powdery mildew including mycelia and conidia were observed on the upper leaves, usually developed from bottom tissues to top parts, also present on stems and siliques. Mycelia on leaves were amphigenous and in patches, often spreading to become effused. These typical symptoms were similar to a previous report of powdery mildew on Broccoli raab (Koike and Saenz 1997). Appressoria are lobed, and foot cells are cylindrical with size 18 to 26 × 7 to 10 μm. Conidia are cylindrical and produced singly, with a size of 35 to 50 × 12 to 21 μm and a length : width ratio greater than two (Koike and Saenz 1997). No chasmothecia were observed under the greenhouse conditions. The symptoms and fungal microscopic characters are typical of Pseudoidium anamorph of Erysiphe (Braun 1995). The specific measurements and characteristics are consistent with previous records of Erysiphe cruciferarum Opiz ex L. Junell (Braun and Cook 2012; Vellios et al. 2017). To identify the pathogen, the partial internal transcribed spacer (ITS) region of rDNA of sample CPD-1 was amplified using primers ITS1 and ITS4 (White et al. 1990). The amplicons were sequenced, and the resulting 559-bp sequence was deposited in GenBank (CPD-1, Accession number: OK160719). A GenBank BLAST search of the ITS sequences showed an exact match (100% query cover, E-value 0, and 100% identity 559/559 bp) with those of E. cruciferarum on hosts Brassica sp. (KY660929.1), B. juncea from Vietnam (KM260718.1) and China (KT957424.1). A phylogenetic tree was generated with the CPD-1 ITS sequence with several of ITS sequences of close species with different hosts obtained from the GenBank. Isolate CPD-1 was grouped with pathogens from Brassica hosts rather than the holotype strain (KU672364.1) from papaveraceous hosts. To fulfill Koch's postulates, pathogenicity was confirmed through inoculation by dusting conidia onto leaves of seven healthy, potted, 14-day-old C. sativa seedlings (cv. Suneson). Seven non-inoculated plants served as a control treatment. The plants were incubated in a greenhouse with a temperature of 18°C (night) to 22°C (day). The inoculated plants developed similar symptoms after 7 days, whereas the control plants remained symptomless. The fungus on the inoculated plants was morphologically identical to that was originally observed on the diseased plants. Though many Brassica spp. have been known to be infected by E. cruciferarum throughout the world, powdery mildew of C. sativa cultivar Crantz in natural conditions by E. cruciferarum has been reported only in the province of Domokos in Central Greece (Vellios et al. 2017). To our knowledge, this is the first report of powdery mildew caused by E. cruciferarum on C. sativa in Montana. Though the powdery mildew on C. sativa was observed in the greenhouse conditions in this work, it poses a potential threat to the production of this biofuel crop in the northwest of the United States.

Novel Drexlerviridae bacteriophage KMI8 with specific lytic activity against Klebsiella michiganensis and its biofilms

The bacterial genus Klebsiella includes the closely related species K. michiganensis, K. oxytoca and K. pneumoniae, which are capable of causing severe disease in humans. In this report we describe the isolation, genomic and functional characterisation of the lytic bacteriophage KMI8 specific for K. michiganensis. KMI8 belongs to the family Drexlerviridae, and has a novel genome which shares very little homology (71.89% identity over a query cover of only 8%) with that of its closest related bacteriophages (Klebsiella bacteriophage LF20 (MW417503.1); Klebsiella bacteriophage 066039 (MW042802.1). KMI8, which possess a putative endosialidase (depolymerase) enzyme, was shown to be capable of degrading mono-biofilms of a strain of K. michiganensis that carried the polysaccharide capsule KL70 locus. This is the first report of a lytic bacteriophage for K. michiganensis, which is capable of breaking down a biofilm of this species.

Konjac anthracnose is caused by Colletotrichum gloeosporioides in Yunnan Province of China

Konjac (Amorphophallus konjac) is an economically important traditional crop in Fengqing County, Yunnan Province, China. Anthracnose symptoms were observed on this crop in June and July of 2020. The plants developed round, oval, or irregular leaf spots with brown edges and central taupe spots, and exhibited serious defoliation and tree weakness. Disease incidence in affected fields reached up to 35%. Anthracnose caused significant economic losses in konjac production and became a limiting factor of the konjac industry in Fengqing County. To date, no control measures of konjac anthracnose have been reported and tested in China. To determine the causal pathogen, symptomatic leaves were collected and cut into 5 mm2 pieces. The leaf peces were surface sterilized in 70% ethanol for 10 s, followed by treatment with 0.1% mercuric chloride for 3 min and three rinses in sterile distilled water. The tissue pieces were transferred onto potato dextrose agar (PDA) and incubated at 28°C. After 4 days of incubation, hyphal tips from leaf pieces were transferred to new PDA to generate pure cultures. The hyphae were initially white, and then became dark green; red-orange conidial masses were observed on the mycelium plate surface at a growth rate of 13.14 mm/day. The conidia, observed under 400× magnification, were colorless, long-oval to fusiform, one-celled, and 15.4 to 18.2 × 3.3 to 5.9 μm in size. To identify the isolate, the genomic DNA of the pathogen was extracted using the CTAB method. The internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and β-tubulin (TUB) gene were PCR amplified using the ITS1/ITS4, GDF/GDR, and Bt2a/Bt2b primer pairs, respectively (Lee et al. 2020). BLASTn search of the obtained 536 bp ITS fragment (GenBank accession no. MT785772), 229 bp GAPDH sequence (MW187543), and 717 bp TUB sequence (MW187544) revealed a 99.44% to 99.63% sequence homology (100% query cover) with ITS (JQ005152, 99.44%), GADPH (JQ005239, 99.63%), and TUB (JQ005587, 99.60%) sequences of the C. gloeosporioides type strain CBS 112999, respectively. The highest homology with other Colletotrichum species was only 98.16%, including C. siamense, the causal agent of anthracnose in A. paeoniifolius and A. konjac (Prasad et al. 2017; Wu et al. 2020). To complete Koch’s postulates, leaves of 3-month-old konjac plants grown in the field were sprayed with a conidial suspension (106 spores/ml) of the isolate YNFQ-1 (sterile water was used as a negative control). Approximately 5 days after inoculation with YNFQ-1, symptoms similar to those in natural conditions appeared, whereas the negative control plants and fruits inoculated with the sterile water had no disease. The pathogen was re-isolated (strain YNFQ-1) from inoculated leaf tissues, and its identity was confirmed with both morphological and molecular (DNA sequences) tools, thus fulfilling Koch's postulates. The culture properties, morphological characteristics, and molecular identification confirmed the identity of the pathogen as C. gloeosporioides. There have been many reports about anthracnose of Amorphophallus; C. siamense causes anthracnose on A. paeoniifolius in India (Prasad et al. 2017) and A. konjac in Hubei, China (Wu et al. 2020), and C. gloeosporioides causes anthracnose of A. muelleri in Yunnan, China (Yang et al. 2020). To the best of our knowledge, this is the first report of C. gloeosporioides causing anthracnose on A. konjac in Fengqing County, China. The results are expected to have important implications in the diagnosis, control, and future research of anthracnose on A. konjac.

ISOLASI DAN IDENTIFIKASI BAKTERI PENAMBAT NITROGEN UNTUK PEMBUATAN BIOFERTILIZER

Nitrogen is a macro nutrient needed by plants. Generally, people use inorganic fertilizers to fulfill nitrogen nutrients in plants. The problem then is, the continuous use of synthetic nitrogen fertilizers has a direct negative impact on the soil and a derivative impact on human health. The use of microorganisms, in this case bacteria, to provide nitrogen to plants can be done by isolating it and making it a biological fertilizer agent. Nitrogen fixing bacteria was isolated on the land of the oil palm plantation of PT Astra Agro Lestari. The isolated nitrogen-fixing bacteria were then tested quantitatively for their ability to fix nitrogen. The bacteria with the highest nitrogen fixing ability were then identified by sequencing their DNA nucleotide bases so that the bacterial strains were identified. The result is that there are 13 bacteria that are able to fix nitrogen with the codes J1, J3, Q5, L1, L11, J31, D1, M6, M5, R1, P2, J4 and C7. The quantitative test shows that bacteria with code D1 are the best at fixing nitrogen in the form of NH4, namely 0.27 ppm. The results of D1 bacterial DNA nucleotide base sequencing showed that the putitive Bacillus aerius strain 24K with identical values ​​and query cover reach

First Report of Bacterial Soft Rot Disease on Pak Choi (Brassica rapa subsp. chinensis) caused by Pectobacterium brasiliense in the United States

Pak choi (Brassica rapa subsp. chinensis) is an important vegetable crop native to China, known for high water content and low caloric value, containing high quality of protein, carbohydrates, fiber, vitamins, minerals, and secondary plant metabolites (Acikgoz, 2016). A pak choi field (8,000 sq. ft.) on Oahu, Hawaii, was visited in May 2020. About 10% plants were infected and showed characteristic symptoms of soft rot, wet lesions, macerated infected stem and necrotic leaves (Figure1A-D); leading to the suspect of one of the most devastating bacterial pathogens within genus Pectobacterium (Boluk et al. 2020; Li et al. 2019; Arizala et al. 2020; Arizala and Arif, 2019). Four infected plants were collected from the field, and stems were surface sterilized with 0.6% sodium hypochlorite solution for 30 sec, followed by three consecutive rinses in distilled water. The stems were aseptically macerated, streaked on Crystal violet pectate medium (CVP) (Hélias et al. 2011), and incubated for 48 h at 26 ± 2°C. The peculiar morphological characteristic of pectolytic bacterial pathogen, forming pits on CVP, were observed (Meng et al. 2016) (Figure 1E). Purification of bacterial colonies were done by re-streaking of a single colony on dextrose peptone agar (DPA—without tetrazolium chloride; Norman and Alvarez 1989). DNA was isolated from bacterial cultures using the DNeasy Blood and Tissue Kit (Qiagen, Germantown, MA), respectively. Molecular identification of four strains (PL243-246) were performed by the sequencing region of the housekeeping gene dnaA (chromosomal replication initiation protein) using Pec. dnaA-F1/R1 primer set (Dobhal et al. 2020). The amplified PCR product was enzymatically cleaned using ExoSAP-ITTM (Affymetrix Inc, Santa Clara, CA), and sent for sequencing at the GENEWIZ facility (Genewiz, La Jolla, CA) using both forward and reverse primers. The dnaA gene sequences were aligned using Geneious, and manually edited to remove the errors. The consensus sequences were analyzed with the NCBI BLASTn tool and were deposited in the NCBI GenBank under the accession numbers MT899920-MT899923. The NCBI BLASTn report indicated that all the sequences shared 99-100% identity and query cover with Pectobacterium brasiliense accession numbers MN544627-29. A phylogenetic analysis, using Geneious, was performed with the dnaA sequences representing different Pectobacterium spp., all strains grouped within the clade of P. brasiliense (Figure 2; Arizala et al, 2020). A pathogenicity assay was carried out in three replications on pak choi grown in pots containing commercial pot mixture, and maintained in the controlled-greenhouse (temperature 26-30°C; relative humidity 50-58%). Three-weeks old plant stems were artificially inoculated with 100 µl bacterial suspensions of PL243 (1.3x 10⁸ CFU/ml), PL244 (1.2x 10⁸ CFU/ml), PL 245 (1.2x 10⁸ CFU/ml) and PL246 (1.1x 10⁸CFU/ml); control plants were inoculated with 100 µl of distilled water (Figure 1F). Two days after inoculation, the soft rot and wilting symptoms (Figure 1G-H), similar to the ones observed on the field, were developed for all four strains tested. Bacteria was successfully re-isolated from the inoculated plants; DNA was isolated, amplified, sequenced for dnaA region and analyzed for 100% homology with original strains, to fulfill Koch’s postulates. Based on the molecular characteristics re-isolates were identical to the original strains. To the best of our knowledge, this is the first report of P. brasiliense on pak choi in the USA. Recent reports indicated that the pathogen could potentially pose a threat to cruciferous crops, therefore, highlighting a need to conduct a state-wide survey for pectinolytic bacteria, and implement better management strategies to combat the vegetable crop losses.

First Report of the Rust Disease Caused by Puccinia crepidis-japonicae on New Host Sonchus arvensis from Pakistan.

Sonchus arvensis (Asteraceae) is a traditional medicinal herb. The aerial parts are a rich source of vitamins, essential amino acids, and minerals, which may help in treatment of fever, inflammation, detoxication, and blood circulation (Li et al. 2018). In October 2018, typical rust symptoms were observed on S. arvensis leaves and stems in Buner district, Malakand division, Khyber PakhtunKhwa Province, Pakistan (34.39°N; 72.61°E). Almost 40% of leaves and stems of five S. arvensis plants displayed severe rust. The specimens were examined microscopically and compared with type specimen description in published literature (Dietel 1908; Hiratsuka et al. 1992). The fungus was identified as Puccinia crepidis-japonicae based on the characteristic of spore morphology and phylogenetic study based on the internal transcribed spacer (ITS) and large subunit (LSU) sequence data. Uredinia were amphigenous, rounded, or somewhat elliptical, naked, small patches, equally spread, brown. Urediniospores (n=30) were globose to ellipsoid, brownish yellow and measured 22.4–24.7 × 20.2–22.1 µm. Urediniospore walls were brownish orange and finely echinulate and 1.7 to 2.1 µm thick with 2 to 3 germ pores. Telia were amphigenous, rounded or elliptic, scattered, dark brown to blackish. Teliospores (n=30) were ellipsoid, subglobose or long ellipsoid, rounded at both ends, not thickened at apex, warted, reddish brown, and measured 31.3–39 × 24.6–26.8 µm. Teliospore walls were reddish-black and about 1.5–2.5 µm thick, and the pedicles were short, hyaline, fragile, become tapered toward apex, and measured 14.4–18.7 × 4.7–9 µm. DNA was extracted from urediniospores, and the combined region of ITS and LSU (28S) were amplified using Rust2Inv (forward primer) and LR6 (Reverse primer) according to the protocol outlined by Aime (2006). A BLASTn search (http://www.ncbi.nlm.nih.gov) showed that the combined ITS and LSU region shared 99% identity (792/804 bp) to the P. crepidis-japonicae accessions (KY798395 from Hawaii, USA) with 100% query cover. The resulting sequence was deposited in GenBank (Accession No. MN093335). Both morphological and molecular characteristics indicatethat this species was P. crepidis-japonicae. To test pathogenicity and fulfill the Koch's postulates, a urediniospore suspension (1 × 104 spores/ml) was sprayed on three 6-week-old plants of S. arvensis, and one as negative control, incubated at 22–24°C. Uredinia were observed on the leaves after 10 days of inoculation, whereas the control plants remained symptomless. Microscopic examination confirmed that the symptoms on plants obtained from the field and greenhouse inoculations were morphologically identical. This fungus has been observed previously on Crepis japonica in China, Hong Kong, Japan, Korea, and Taiwan, on Prenanthes spp. in China and on Youngia tenuifolia and Y. fusca in China (Farr and Rossman 2021). Pereira et al. (2002) suggested that P. crepidis-japonicae may play a significant role as a biocontrol agent against its weed host. To the best of our knowledge, there are no other reports of this fungus on any other hosts in Pakistan. The specimen has been vouchered in LAH Herbarium, Department of Botany, University of the Punjab, Lahore, Pakistan (LAH36343). This is the first report of P. crepidis-japonicae on S. arvensis as a new host from Pakistan.

Analisis sekuen mtDNA COI Pari Totol Biru yang didaratkan di Tempat Pendaratan Ikan Kota Tarakan

Identifikasi spesies berbasis sekuen mtDNA COI telah diaplikasikan ke berbagai tujuan, termasuk untuk melindungi biodiversitas dan eksploitasi sumberdaya ikan yang berlebihan. Tujuan penelitian ini adalah untuk menganalisis kualitas sekuen gen mtDNA COI pari totol biru yang diperoleh dari tempat pendaratan ikan di Kota Tarakan. Hasil PCR mtDNA sampel diperoleh 456.1 ng/µL dengan rasio A260/280 yaitu 1.93 dan pita tunggal dengan ukuran sekitar 700 bp. Proses sekuensing dilakukan dengan metode bi-directional sequencing. Hasil sekuensing tersebut dianalisis dengan menggunakan aplikasi sequence scanner v.2.0. dan diperoleh data persentase pure base kategori rendah (low) dan sedang (medium) melebihi 10% yang menjelaskan sekuen konsensus berkualitas rendah. Hasil ini berkorelasi dengan analisis BLAST yang menunjukkan perolehan parameter query cover hanya sebesar 23% saja. Meskipun nilai kemiripan sekuen sampel dengan Neotrygon kuhlii voucher BW-A2578 sebesar 98.28%, hasil ini memiliki validitas yang rendah karena tidak didukung dengan nilai query cover yang memadai.

The First Report of Orobanche laxissima Parasitizing Pomegranate (Punica granatum) in Georgia

Orobanche laxissima Uhlich & Rätzel (Orobanchaceae) is a polyphagous root parasitic plant distributed in the Caucasus Mountains and Transcaucasia; especially Russia, Georgia, Armenia, Azerbaijan, N.E. Turkey (Piwowarczyk et al. 2019). It infects many wild or sometimes cultivated trees and shrubs, such as Betulaceae, Oleaceae, Fagaceae, Aceraceae, Cornaceae, usually Fraxinus L., Fagus L., Carpinus L. (Piwowarczyk et al. 2019, 2020). Punica granatum L. (Lythraceae), commonly known as pomegranate, is native to the Caucasus, the Himalayas in North Pakistan and Northern India, and is widely cultivated, e.g. in USA and throughout the Mediterranean. Pomegranate is one of the first domesticated fruits and have been used in folk medicine or as a food for centuries. Fruit, seed, leaves, flower, root, or barks extracts have extensive medicinal properties (Shaygannia et al. 2015). Field surveys conducted in south-eastern Georgia in May 2019 revealed extensive infestations of O. laxissima on the roots of P. granatum in one locality in Kakheti Province, near Sighnaghi (41°37,4 N, 45°56,3 E, 480 m elevation), in roadside or hills scrub and cultivated areas of pomegranate. The infection was confirmed by verifying the attachment of the Orobanche to the Punica root. The population of the parasite consisted of at least a ca. thousand shoots, sometimes in one clump was ca. 100 individuals. A single plant of pomegranate was parasitized by few to c.a. a hundred of broomrape plants, and 10 to 20% of the ca. 1 ha location was infested. The main botanical features of the O. laxissima are: i) stem simple, (10–)25–40(–100) cm high, with haustoria; ii) inflorescence usually long to short cylindrical or lax, usually many-flowered; iii) calyx-segments entire or bidentate, rarely with 4 teeth; iv) corolla (16–)22–24(–31) mm long, tubular-bell-shaped; purple, pink, rarely dirty yellow, light brown; v) stigma purple, orange, or yellow (Piwowarczyk et al. 2019, 2020). For molecular analysis, total genomic DNA was extracted from the sample and the plastid gene rbcL (rubisco large subunit) was sequenced and amplified as described in Piwowarczyk et al. (2015). The sequence (1231 bp) was deposited in GenBank (MN384886). BLAST search found that it was most similar to (Query Cover 100%, Per Ident. 100%) O. laxissima (KR260928). To the best of our knowledge, this is the first report of a O. laxissima parasitizing P. granatum. O. laxissima appearing in large numbers on singles pomegranate shrubs can weaken the plants, and reduce flowering and fruiting. In the Caucasus region, O. laxissima was observed in mesophilic forests and shrubs, but our report suggests the possibility of a potential spread to neighboring cultivated areas, especially fruit trees and shrubs. Until now, only one report of pathogenic plants was documented for P. granatum, included Phelipanche aegyptiaca (Pers.) Pomel and O. crenata Forssk. in Israel (Dor et al. 2014).

CHARACTERIZATIONS OF Trichoderma sp. AND ITS EFFECT ON Ralstonia solanacearum OF TOBACCO SEEDLINGS

This study aims to determine the molecular-based characteristics of Trichoderma sp. Tc-Jjr-02 and its effect as a biocontrol agent in protecting tobacco seedlings against bacterial wilt caused by R. solanacearum. The characterization of biocontrol agents was based on morphological and molecular data’s observation using microscope and the key of determination. The in vivo experiments was consist of five treatments: (1) inoculation of Trichoderma isolates at six hours before R. solanacearum inoculation, (2) inoculation of Trichoderma isolates at six hours after R. solanacearum inoculation (3) simultaneous inoculation of Trichoderma isolates and R. solanacearum, (4) inoculated only with R. solanacearum, and (5) without any inoculation. The experiment was repeated six times. Based on BLAST’s analysis, the Tc-Jjr-02 sequence is in accordance with T. asperellum with 100% Query Cover. Inoculation of T. asperellum Tc-Jjr-02 at six hours before and after and simultaneously with pathogens providing protection for young tobacco plants by slowing down the time for the onset of blight by 100–162%, reducing the symptom index by 56–63%, and increasing the dry weight of plant biomass by 39–53% compared to tobacco seeds which were only inoculated with R. solanacearum.