In vitro Inoculation of Fresh or Frozen Rumen Fluid Distinguishes Contrasting Microbial Communities and Fermentation Induced by Increasing Forage to Concentrate Ratio

In vitro rumen batch culture is a technology to simulate rumen fermentation by inoculating microorganisms from rumen fluids. Although inocula (INO) are commonly derived from fresh rumen fluids, frozen rumen fluids are also employed for the advantages of storing, transporting, and preserving rumen microorganisms. The effects of frozen INO on microbial fermentation and community may be interfered with by substrate type, which has not been reported. This study was designed to test whether rumen fluid treatments (i.e., fresh and frozen) could interact with incubated substrates. A complete block design with fractional arrangement treatment was used to investigate the effects of INO (fresh or frozen rumen fluids) and concentrate-to-forage ratios (C/F, 1:4 or 1:1) on rumen fermentation and microbial community. The effects of increasing C/F were typical, including increased dry matter (DM) degradation and total volatile fatty acids (VFA) concentration (P < 0.001), and decreased acetate to propionate ratio (P = 0.01) and bacterial diversity of richness and evenness (P ≤ 0.005) with especially higher fermentative bacteria such as genus Rikenellaceae_RC, F082, Prevotella, Bacteroidales_BS11, Muribaculaceaege, and Christensenellaceae_R-7 (P ≤ 0.04). Although frozen INO decreased (P < 0.001) DM degradation and altered rumen fermentation with lower (P ≤ 0.01) acetate to propionate ratio and molar proportion of butyrate than fresh INO, typical effects of C/F were independent of INO, as indicated by insignificant INO × C/F interaction on substrate degradation, VFA profile and bacterial community (P ≥ 0.20). In summary, the effect of C/F on fermentation and bacterial diversity is not interfered with by INO type, and frozen INO can be used to distinguish the effect of starch content.

A rapid, simple, and highly efficient method for VIGS and in vitro-inoculation of plant virus by INABS applied to crops that develop axillary buds and can survive from cuttings

Background Virus-induced gene silencing (VIGS) is one of the most convenient and powerful methods of reverse genetics. In vitro-inoculation of plant virus is an important method for studying the interactions between viruses and plants. Agrobacterium-based infiltration has been widely adopted as a tool for VIGS and in vitro-inoculation of plant virus. Most agrobacterium-based infiltration methods applied to VIGS and virus inoculation have the characteristics of low transformation efficiencies, long plant growth time, large amounts of plant tissue, large test spaces, and complex preparation procedures. Therefore, a rapid, simple, economical, and highly efficient VIGS and virus inoculation method is in need. Previous studies have shown that the selection of suitable plant tissues and inoculation sites is the key to successful infection. Results In this study, Tobacco rattle virus (TRV) mediated VIGS and Tomato yellow leaf curl virus (TYLCV) for virus inoculation were developed in tomato plants based on the agrobacterium tumefaciens-based infiltration by injection of the no-apical-bud stem section (INABS). The no-apical-bud stem section had a “Y- type” asymmetric structure and contained an axillary bud that was about 1–3 cm in length. This protocol provides high transformation (56.7%) and inoculation efficiency (68.3%), which generates VIGS transformants or diseased plants in a very short period (8 dpi). Moreover, it greatly reduces the required experimental space. This method will facilitate functional genomic studies and large-scale disease resistance screening. Conclusions Overall, a rapid, simple, and highly efficient method for VIGS and virus inoculation by INABS was developed in tomato. It was reasonable to believe that it can be used as a reference for the other virus inoculation methods and for the application of VIGS to other crops (such as sweet potato, potato, cassava and tobacco) that develop axillary buds and can survive from cuttings.

Identification and Isolation Pattern of Globisporangium spp. from a Sanionia Moss Colony in Ny-Ålesund, Spitsbergen Is., Norway from 2006 to 2018

Globisporangium spp. are soil-inhabiting oomycetes distributed worldwide, including in polar regions. Some species of the genus are known as important plant pathogens. This study aimed to clarify the species construction of Globisporangium spp. and their long-term isolation pattern in Sanionia moss in Ny-Ålesund, Spitsbergen Is., Norway. Globisporangium spp. were isolated at two-year intervals between 2006 and 2018 at a Sanionia moss colony, Ny-Ålesund, Spitsbergen Is., Norway. The isolates were obtained by using three agar media and were identified based on sequences of the rDNA-ITS region and cultural characteristics. Most of the Globisporangium isolates obtained during the survey were identified into six species. All six species were grown at 0 °C on an agar plate and used to infect Sanionia moss at 4 and/or 10 °C under an in vitro inoculation test. The total isolation frequency of Globisporangium gradually decreased throughout the survey period. The isolation frequency varied among the six species, and four of the species that showed a high frequency in 2006 were rarely isolated after 2016. The results suggested that Globisporangium inhabiting Sanionia moss in Ny-Ålesund has a unique composition of species and that most of the species reduced their population over the recent decade.

CMSERK GENE IS EXPRESSED IN RESPONSE TO THE ATTACK OF PATHOGEN FUNGI COLLETOTRICHUM ACUTATUM AND BOTRYTIS CINEREA

The genus Cattleya groups orchids originate in tropical zones of South and Central America. One of the most representative species of ornamental importance is Cattleya maxima Lindl. In this study the fungal pathogens Colletotrichum acutatum and Botrytis cinerea were isolated and their pathogenicity was determined by in vitro inoculation of Cattleya maxima. Pathogenicity tests resulted positive for infection with C. acutatum after seven days of inoculation while as for B. cinerea the symptoms of infection appeared after two days. Quantitative PCR revealed that CmSERK gene is more expressed in tissue under fungal attack. These results suggest that CmSERK gene plays an important role in the activation of defense-related responses.

Response of citrus hybrids to Alternaria alternata inoculation

Citrus orchards have some limitations, such as the occurrence of phytosanitary problems. Alternaria brown spot (ABS) is caused by fungus Alternaria alternata, which affects several parts of the plant by producing a host-specific toxin, known as ACT. ABS is a limiting factor in orchards due to the susceptibility of most planted cultivars: ‘Murcott’ tangor and ‘Ponkan’ tangerine. The selection of varieties resistant/tolerant to the disease has economic importance. Therefore, the aim of this experiment was to evaluate the response to A. alternata inoculation in a population of ‘Murcott’ tangor vs ‘Pera’ sweet orange hybrids. Leaves of 2-3 centimeters in length of ‘Murcott’ tangor, ‘Pera’ sweet orange, ‘Ponkan’, ‘Dancy’, ‘Fremont’ tangerine and 198 hybrids were collected. For in vitro inoculation, monosporic A. alternata culture at concentration of 105 conidia mL-1 was used. Inoculated leaves were stored in humid chamber. After 24, 48 and 72 hours of inoculation, leaf lesions were evaluated following a diagrammatic scale. The results obtained showed that most hybrids from the crossing of ‘Murcott’ tangor vs ‘Pera’ sweet orange are susceptible to ABS. However, 44 are resistant and ten are tolerant. Among ABS-tolerant hybrids, some have phenotype similar to that of cultivated and commercialized hybrids.

IN VITRO COMPATIBILITY BETWEEN INSECTICIDES AND THE COMMERCIAL BIOINSECTICIDE AGREE® WG

Compatibility studies are essential for the integration and simultaneous use of chemical and biological pest control methods since they are necessary for an Integrated Pest Management (IPM) program. In this work, the aim was to evaluate the compatibility of insecticides used in soybean and cotton crops for pest control with Bacillus thuringiensis (Bt). The in vitro inoculation technique was used with B. thuringiensis var. kurstaki and B. thuringiensis var. aizawai, in culture medium containing the following insecticides: beta-cyfluthrin (Bulldock®), methomyl (Bazuka®), thiamethoxam + lambda-cialotrina (Engeo Pleno®), zeta-cypermethrin (Fury 200®), acetamiprid (Saurus®), bifenthrin + carbosulfano (Talisman®) and bifenthrin (Talstar®), in Petri dishes. The Petri dishes were taken to the B.O.D. (Biological Oxygen Demand), at a temperature of 30 ± 1 ºC, 70 ± 10% RH (relative humidity) and a photophase of 12 h, for 24 hours. Colony growth was measured, and Colony Forming Units (CFU) counted in the total area of the Petri dish. The product that allowed growth to be significantly equal to or higher than the control was established as compatible, and the one that did not allow growth or was significantly less than the control was incompatible. It was found that all insecticides were classified as incompatible with the bioinsecticide.

Synergistic Antifungal Activity of Green Synthesized Silver Nanoparticles and Epoxiconazole against Setosphaeria turcica

It is urgent to develop highly efficient and eco-friendly antimicrobial agents for integrated control of phytopathogens. Silver nanoparticles (AgNPs) were synthesized by Ligustrum lucidum leaf extract. UV-vis spectrum showed that there was a strong absorbance at 438 nm. Transmission electron microscopy (TEM) images displayed that synthesized nanoparticles were near spherical with an average size of 13 nm. The antimicrobial effect of AgNPs was evaluated through methods of paper disk diffusion, colony growth, conidia germination, and in vitro inoculation. The 50% inhibition concentration (IC50) of AgNPs against Setosphaeria turcica was 170.20 μg/mL calculated by SPSS 13.0. In addition, it displayed a significant synergistic antifungal effect when AgNPs were combined with epoxiconazole at the ratios of 8 : 2 and 9 : 1. The results of this study provide a novel fungistat not only for comprehensive control of plant fungi but also for reducing chemical pesticides use and avoiding drug-resistant phytopathogen generation.

In vitro growth response to bacterial wilt pathogen of banana (var. lakatan, Musa acuminata Colla) plantlets regenerated from ethyl methanesulfonate-treated shoot explants

Bacterial wilt caused by Ralstonia solanacearum leads to death of infected suckers and reduces the yield of commercially important banana varieties like Lakatan. Among the many varieties of banana, no germplasm with bacterial wilt resistance has been identified yet (Tripathi et al 2004). Mutation induction in plants to develop disease resistance genes using physical or chemical mutagens has been used as alternative to harmful pesticides. To induce mutation for the possible development of resistance to bacterial wilt, shoot tips of Stage 2 in vitro-grown Lakatan plantlets were exposed to 0.1% and 0.2% ethyl methanesulfonate (EMS) for 12 and 24h. Treated and untreated explants were cultured in-vitro to regenerate plantlets. Shoots emerged two days after in vitro inoculation of explants treated with – 0.1% EMS for 12h. Significantly longer shoots also developed from the cultures compared to the untreated explants. The other explants exposed to other treatments had shoot emergence one to three days later. Falcate, curled, irregularly-shaped, and yellowish leaves and pseudostems also developed in EMS-treated cultures. Untreated plantlets exhibited at least one bacterial wilt symptom such as leaf spots, necrosis at pseudostem base, and death six days from the introduction of Ralstonia solanacearum in vitro. Plantlets from explants exposed to 0.1% EMS for 12h did not exhibit disease symptoms even after ten days of exposure to the pathogen and had 100% survival. Seventy one percent of plantlets from explants exposed to 0.1% EMS for 24h and 55% from explants treated with 0.2% EMS for 24h also survived without infection. The surviving plantlets need to be studied further for their ex vitro responses to the pathogen and determine possible genetic changes due to the chemical mutagen treatment.