Discovery of microRNA-like Small RNAs in Pathogenic Plant Fungus Verticillium nonalfalfae Using High-Throughput Sequencing and qPCR and RLM-RACE Validation

Verticillium nonalfalfae (V. nonalfalfae) is one of the most problematic hop (Humulus lupulus L.) pathogens, as the highly virulent fungal pathotypes cause severe annual yield losses due to infections of entire hop fields. In recent years, the RNA interference (RNAi) mechanism has become one of the main areas of focus in plant—fungal pathogen interaction studies and has been implicated as one of the major contributors to fungal pathogenicity. MicroRNA-like RNAs (milRNAs) have been identified in several important plant pathogenic fungi; however, to date, no milRNA has been reported in the V. nonalfalfae species. In the present study, using a high-throughput sequencing approach and extensive bioinformatics analysis, a total of 156 milRNA precursors were identified in the annotated V. nonalfalfae genome, and 27 of these milRNA precursors were selected as true milRNA candidates, with appropriate microRNA hairpin secondary structures. The stem-loop RT-qPCR assay was used for milRNA validation; a total of nine V. nonalfalfae milRNAs were detected, and their expression was confirmed. The milRNA expression patterns, determined by the absolute quantification approach, imply that milRNAs play an important role in the pathogenicity of highly virulent V. nonalfalfae pathotypes. Computational analysis predicted milRNA targets in the V. nonalfalfae genome and in the host hop transcriptome, and the activity of milRNA-mediated RNAi target cleavage was subsequently confirmed for two selected endogenous fungal target gene models using the 5′ RLM-RACE approach.

Isolation of Pseudomonas Species from Rhizospheric Soil and its Antagonistic Effect on Plant Pathogen

The pathogen attack on plants (such as tubers and small plants) has substantially progressed by 60% irrespective of even when the plants have strong immune system for the past five decades. It drastically affects the plant growth, yield and production. Certain pesticides and fertilizers have been tried to control the progressing pathogenic microorganisms. Although these measures have proved worthless due to the resistance shown by those pathogens and resulted in environmental pollution over a period of time. Bio control measures are well appreciated as it is ecofriendly. Antagonist microorganism (bacteria, fungi) will be used to suppress the growth of the invading pathogens. In this study, Pseudomonas species , were screened from rhisozpheric soill from the local region of Srivilliputhur. Eleven isolates from rhizospheric soil were screened and characterized by Gram staining, catalase test, Voges-Proskauer test, and oxidase test. The morphological studies of all the screened Pseudomonas isolates were observed as Gram negative. Further, all the isolates were found to be catalase positive and negative for VP test. The isolates were subjected to antagonistic effect on plant fungal pathogen such as Rhizoctonia solani. Among eleven Pseudomonas isolates four isolates showed antagonistic effect against R.solani. In the present investigation, four isolates showed potent antagonistic effect and could be used as effective bio-control agent against plant fungal pathogen R.solani.

Phytochemical Investigation, Antimicrobial, Antioxidant and Anticancer Activities of Acer cappadocicum Gled

The appearance of novel microbial resistance, diverse cancer ailment and several other morbidities such as appetite loss, hair loss, anemia, cell damage, etc., are among most critical situation that keeps the phytochemical quest on. Thus, this study characterized the antimicrobial, antioxidant, and anticancer potentials of a rarely accessed Acer cappadocicum gled (AC) population thriving in a remote Palas Valley in northern Pakistan. Leaf extracts of the plant were prepared in organic solvents with different polarities through maceration. Extracts were subjected to antimicrobial, antioxidant, and anticancer activities using agar well, DPPH and cell viability assays. A. cappadocicum methanolic extract (ACM) significantly inhibited bacterial growth, followed by n-butanolic extract (ACB) with the second-highest bacterial inhibition. Similar activity was observed against mycelial growth inhibition in plant-fungal pathogen by ACM and ACB. However, human pathogenic fungi did not affect much by extracts. In antioxidant assessment, the chloroform extract (ACC) showed strong scavenging activity and in cytotoxic evaluation, extracts restricted growth proliferation in cancer cells. The inhibitory evidence of extracts, potent scavenging ability, and low cell viability of human-derived cell lines supports the antimicrobial, antioxidant and anticancerous potential of A. cappadocicum. It advances our quest for natural product research.

Ceratocystis fimbriata Employs a Unique Infection Strategy Targeting Peltate Glandular Trichomes of Sweetpotato (Ipomoea batatas) Plants

The infection processes of Ceratocystis fimbriata BMPZ13 (BMPZ13) was elucidated on vegetative tissues of sweetpotato plants employing light and scanning electron microscopy. Vegetative tissues infected with C. fimbriata BMPZ13 by either wounding or nonwounding inoculation methods developed typical disease symptoms, establishing black rot in stems and necrosis on buds, young leaves, and stems of sprouts, in addition to wilt on leaves and shoot cuttings, typical of vascular associated diseases. The runner hyphae of C. fimbriata BMPZ13 formed from germinated conidia were able to directly penetrate the epidermal cuticle for initial infection and invade sweetpotato peltate glandular trichomes, specialized secretory structures to store and secrete metabolites. A two-step biotrophic phase was observed with nonwounding inoculation on leaves and stems, featuring both intercellular and intracellular invasive hyphae, with the latter found within living cells of the leaf epidermis. Subsequent to the biotrophic phase was a necrotrophic phase displaying cell death in infected leaves and veins. Additionally, this cell death was an iron-associated ferroptosis, supporting the notion that iron is involved in the necrotrophic phase of C. fimbriata BMPZ13 infection. Significantly, we establish that C. fimbriata employs a unique infection strategy: the targeting of peltate glandular trichomes. Collectively, our findings show that C. fimbriata is a plant fungal pathogen with a hemibiotrophic infection style in sweetpotato vegetative tissues.

Tritrophic Interactions Between an Invasive Weed (Lepidium latifolium), an Insect Herbivore (Bagrada hilaris), and a Plant Pathogenic Fungus (Albugo lepidii)

Perennial pepperweed, Lepidium latifolium L. (Brassicales: Brassicaceae), is an invasive weed that can form dense stands and displace native species. Bagrada hilaris Burmeister (Hemiptera: Pentatomidae) is a serious economic pest of Brassicaceae vegetable crops. Bagrada bug also feeds on L. latifolium and may interact with the plant fungal pathogen Albugo lepidii S.I. (Peronosporales: Albuginaceae) to affect biological control of L. latifolium. A series of laboratory experiments, including Y-tube olfactometer and host-choice tests, were conducted to investigate B. hilaris host-preference behavior. Adults were attracted to the odor of healthy L. latifolium compared with A. lepidii-infected leaves. Bagrada hilaris consistently preferred to feed on healthy L. latifolium when offered both healthy and A. lepidii-infected plants. Experiments were conducted to determine the effects of A. lepidii-infected L. latifolium on B. hilaris survival and development. Survival of all B. hilaris immature stages and adults was markedly reduced for those reared on A. lepidii-infected leaves. Total development time and stage-specific development were faster on healthy L. latifolium leaves compared with A. lepidii-infected leaves. In addition, the ability of B. hilaris adults to passively transmit the rust was studied. Our data demonstrated that B. hilaris could acquire the rust spores while feeding, but it did not passively transmit the pathogen to healthy plants.