The yeast-like fungus Aureobasidium pullulans PL5 is a microbial antagonist against postharvest pathogens of fruits. The strain is able to produce hydrolases, including glucanases, chitinases and ...proteases. The alkaline serine protease gene ALP5 from A. pullulans was cloned, inserted into the vector pPIC9 to construct pPIC9/ALP5, and then expressed in Pichia pastoris strain KM71. ALP5 had a molecular mass of 42.9kDa after 5days growth with 1% methanol induction at 28°C. The recombinant protease expressed in P. pastoris showed its highest activity under alkaline conditions (at pH10) and a temperature of 50°C. The antifungal activity of the recombinant protease was investigated against Penicillium expansum, Botrytis cinerea, Monilinia fructicola and Alternaria alternata in vitro and on apple. The recombinant protease reduced significantly the spore germination and the germ tube length of the tested pathogens in PDB medium. The highest level of protease efficacy was observed against M. fructicola and B. cinerea, whereas a lower efficacy was observed against P. expansum and A. alternata indicating a possible effect of the pathogen cell wall composition on the proteolytic activity of the recombinant protease. The presence of protease was able to cause the swelling of the hyphae of B. cinerea, under an optical microscope. The recombinant protease expressed in P. pastoris was more active against the pathogens in vitro than the same enzyme expressed in E. coli in previous studies. The efficacy of ALP5 was also evaluated against the pathogens in vivo on cv Golden Delicious apples. The protease was more efficient in controlling M. fructicola, B. cinerea and P. expansum than A. alternata. However, the extent of the activity was dependent on the enzyme concentration and the length of fruit storage. This study demonstrated the capacity of the alkaline serine protease to keep its enzymatic activity for some days in the unfavorable environment of the fruit wounds. The alkaline serine protease could be developed as a postharvest treatment with antimicrobial activity for fruit undergoing a short storage period.
•The alkaline serine protease ALP5 of A. pullulans PL5 was expressed in P. pastoris.•ALP5 reduced significantly spore germination and germ tube length of four pathogens.•ALP5 reduced the rots on apples caused by B. cinerea, M. fructicola and P. expansum.•Its activity was dependent on its concentration, the storage time and the pathogen.•ALP5 is able to keep its enzymatic activity for some days in fruit wounds.
Soil-borne cereal mosaic virus (SBCMV) is a furovirus with rigid rod-shaped particles containing an ssRNA genome, transmitted by
Led., a plasmodiophorid that can persist in soil for up to 20 years. ...SBCMV was reported on common and durum wheat and it can cause yield losses of up to 70%. Detection protocols currently available are costly and time-consuming (real-time PCR) or have limited sensitivity (ELISA). To facilitate an efficient investigation of the real dispersal of SBCMV, it is necessary to develop a new detection tool with the following characteristics: no extraction steps, very fast results, and high sensitivity to allow pooling of a large number of samples. In the present work, we have developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) protocol with such characteristics, and we have compared it with real-time PCR. Our results show that the sensitivity of LAMP and real-time PCR on cDNA and RT-LAMP on crude extracts are comparable, with the obvious advantage that RT-LAMP produces results in minutes rather than hours. This paves the way for extensive field surveys, leading to a better knowledge of the impact of this virus on wheat health and yield.
A 20-year study of a pine stand on post-agricultural land showed that woody debris in the form of organic matter can be successfully used to restore symbiotic mycorrhizal communities, as is the case ...with forest soils. Woody substrates restored organic matter in soils altered by long agricultural use and had a positive effect on the composition of mycobiota antagonistic to pathogens, especially to Heterobasidion annosum, the causal agent of the dangerous disease root and stump rot of many forest tree species, including stands of Pinus sylvestris (L.). In a study that started in 2001 in the forest district of Czarne Człuchowskie (northern Poland), the following organic materials were used: wood residues (W), sawdust (S), bark compost (B), and compost applied to the root zone during planting (G). The organic materials were spread in the form of mulch over the entire area during planting. After twenty years, it was found that the substrates used provided suitable growth conditions for mycobiome useful for pines. The addition of organic matter did not change the alpha biodiversity of the soil, but in the long term led to significant changes in the composition of mycobiota (beta biodiversity). The changes in the soil after the addition of organic material naturally accelerated the formation of the forest habitat. A number of fungi evolved that degraded added lignin and cellulose while being antagonists of H. annosum and other pine pathogens. In particular, the well-known hyperpathogens of the genus Trichoderma played an important role by promoting resistance of the soil environment to pathogens. Soil enrichment by bark compost and wood residues increased the relative abundance of Trichoderma more than fourfold.Mycorrhizal fungi became dominant in soil enriched with organic matter. After enriching the soil with bark compost, the relative abundance of Amphinema and Inocybe increased to 5%. The relative abundance of Russula in soil enriched with wood residues and sawdust increased to 9% and 5%, respectively. Mycorrhizal fungi, e.g., of the genus Ąmanita, Rusula, which formed root mycorrhizae, not only increased the root receiving area many times over, but also protected the roots (mechanically and chemically from pathogens). Altogether, the observed positive changes increase the chances that the first generation of pines will survive on the ground.
Despite advances in science and technology, agricultural yield losses due to plant pathogens and pests currently account for 30% of global production ...
Advances in transient expression technologies have allowed the production of milligram quantities of proteins within a matter of days using only small amounts (tens of grams) of plant tissue. Among ...the proteins that have been produced using this approach are the structural proteins of viruses which are capable of forming virus-like particles (VLPs). As such particulate structures are potent stimulators of the immune system, they are excellent vaccine candidates both in their own right and as carriers of additional immunogenic sequences. VLPs of varying complexity derived from a variety of animal viruses have been successfully transiently expressed in plants and their immunological properties assessed. Generally, the plant-produced VLPs were found to have the expected antigenicity and immunogenicity. In several cases, including an M2e-based influenza vaccine candidate, the plant-expressed VLPs have been shown to be capable of stimulating protective immunity. These findings raise the prospect that low-cost plant-produced vaccines could be developed for both veterinary and human use.
Background
Bakanae
disease, caused by seed-borne
Fusarium
species, mainly
F. fujikuroi
, is a rice disease whose importance is considerably increasing in several rice growing countries, leading to ...incremental production losses.
Results
A germplasm collection of
japonica
rice was screened for
F. fujikuroi
resistance, allowing the identification of accessions with high-to-moderate levels of resistance to
bakanae
. A GWAS approach uncovered two genomic regions highly associated with the observed phenotypic variation for response to
bakanae
infection on the short arm of chromosome 1 (named as
qBK1_628091
) and on the long arm of chromosome 4 (named as
qBK4_31750955
). High levels of phenotypic resistance to
bakanae
were associated to the cumulated presence of the resistant alleles at the two resistance loci, suggesting that they can provide useful levels of disease protection in resistance breeding. A fine comparison with the genomic positions of qBK1_628091 and qBK4_31750955 with respect to the QTLs for
bakanae
resistance reported in the literature suggests that the resistant loci here described represent new genomic regions associated to
F. fujikuroi
resistance. A search for candidate genes with a putative role in
bakanae
resistance was conducted considering all the annotated genes and
F. fujikuroi
-related DEGs included in the two genomic regions highlighting several gene functions that could be involved in resistance, thus paving the way to the functional characterization of the resistance loci.
Conclusions
New effective sources for
bakanae
resistance were identified on rice chromosomes 1 and 4 and tools for resistance breeding are provided.
Tomato (Solanum lycopersicum L.), family Solanaceae, has become in the past fifty years one of the most important and extensively grown horticultural crops in the Mediterranean region and throughout ...the world. In 2019, more than 180 million tonnes of tomato have been produced worldwide, out of which around 42 million tonnes in Mediterranean countries. Due to its genetic properties, tomato is afflicted by numerous plant diseases induced by fungal, bacterial, phytoplasma, virus, and viroid pathogens. Not only is its genetic inheritance of great importance to the management of the numerous tomato pathogens, but equally as important are also the present climate changes, the recently revised phytopathological control measures, and the globalization of the seed industry. Thus, the recognition of symptoms and the knowledge of the distribution and spread of the disease and of the methods for early detection of the pathogens are the major prerequisites for a successful management of the disease. In this review, we will describe the main tomato pathogens in the Mediterranean area that impact mostly the tomato yield and provide the current and perspective measures necessary for their successful management.
A real-time loop-mediated isothermal amplification (LAMP) assay was developed for simple, rapid and efficient detection of the Olea europaea geminivirus (OEGV), a virus recently reported in different ...olive cultivation areas worldwide. A preliminary screening by end-point PCR for OEGV detection was conducted to ascertain the presence of OEGV in Sicily. A set of six real-time LAMP primers, targeting a 209-nucleotide sequence elapsing the region encoding the coat protein (AV1) gene of OEGV, was designed for specific OEGV detection. The specificity, sensitivity, and accuracy of the diagnostic assay were determined. The LAMP assay showed no cross-reactivity with other geminiviruses and was allowed to detect OEGV with a 10-fold higher sensitivity than conventional end-point PCR. To enhance the potential of the LAMP assay for field diagnosis, a simplified sample preparation procedure was set up and used to monitor OEGV spread in different olive cultivars in Sicily. As a result of this survey, we observed that 30 out of 70 cultivars analyzed were positive to OEGV, demonstrating a relatively high OEGV incidence. The real-time LAMP assay developed in this study is suitable for phytopathological laboratories with limited facilities and resources, as well as for direct OEGV detection in the field, representing a reliable method for rapid screening of olive plant material.
Bakanae disease, caused by Fusarium fujikuroi, was investigated under different CO2 and temperature environments in order to simulate climate changes in the F. fujikuroi–rice pathosystem. F. ...fujikuroi‐infected plants were grown under six phytotron conditions: low (18/22 °C night/day), medium (22/26 °C), and high (26/30 °C) temperature, at either ambient (450 ppm) or elevated (850 ppm) CO2 concentrations. Bakanae disease index (DI), seedling death incidence, fungal DNA quantity, and chlorophyll and carbohydrate contents varied significantly in infected plants as a consequence of changes in both CO2 and temperature. Plant height and dry weight were only influenced by single factors (temperature for height, and temperature or CO2 for dry weight), and not by the CO2 × temperature interaction. Medium and high temperatures (irrespective of the CO2 level) increased the DI significantly (range from 67.5% to 95.8%) compared to low temperatures (range from 45.8% to 47.5%). Under elevated CO2 levels, noticeable differences in the expression of four rice defence‐related genes and fungal DNA quantity were observed between those plants grown at higher temperatures and those at lower temperatures. Overall, combined and single effects of elevated CO2 and high temperatures seem to be favourable for bakanae disease development in the Mediterranean basin.
Combined and single effects of elevated CO2 and high temperatures were favourable for bakanae disease development in rice plants. Fungal quantity and plant death incidence varied significantly due to changes in CO2 and temperature.
In the last decades, the evolution of molecular diagnosis methods has generated different advanced tools, like loop-mediated isothermal amplification (LAMP). Currently, it is a well-established ...technique, applied in different fields, such as the medicine, agriculture, and food industries, owing to its simplicity, specificity, rapidity, and low-cost efforts. LAMP is a nucleic acid amplification under isothermal conditions, which is highly compatible with point-of-care (POC) analysis and has the potential to improve the diagnosis in plant protection. The great advantages of LAMP have led to several upgrades in order to implement the technique. In this review, the authors provide an overview reporting in detail the different LAMP steps, focusing on designing and main characteristics of the primer set, different methods of result visualization, evolution and different application fields, reporting in detail LAMP application in plant virology, and the main advantages of the use of this technique.
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