Cloning, characterization, expression and antifungal activity of an alkaline serine protease of Aureobasidium pullulans PL5 involved in the biological control of postharvest pathogens

An alkaline protease gene was amplified from genomic DNA and cDNA of the antagonistic yeast-like fungus Aureobasidium pullulans PL5, a biocontrol agent effective against Monilinia laxa on stone fruit and Botrytis cinerea and Penicillium expansum on pome fruits. An open reading frame of 1248 bp encoding a 415-amino acid (aa) protein with a calculated molecular weight (M_r) of 42.9 kDa and an isoelectric point (pI) of 4.5 was characterized. The cDNAALP5 gene had an 18-amino acid signal peptide, one N-gylcosylation, one histidine active site, and one serine active site. The ALP5 gene with a M_r of 1351 bp contained two introns. One intron was of 54 bp, while the other was of 50 bp. Protein BLAST and phylogenetic tree analysis of the deduced amino sequences from the cDNAALP5 gene showed that the encoded protein had 100% homology to a protease enzyme (ALP2) of a sea strain of A. pullulans, suggesting that the protein ALP5 was an alkaline serine protease. Expression of ALP5 in Escherichia coli BL21 (DE3), followed by identification with Western-blotting, purification with Ni-NTA and analysis of enzymatic activity, yielded an homogeneous recombinant ALP5 which hydrolysed the substrate casein and inhibited the mycelial growth of the pathogens. At its optimal pH of 10.0 and reaction temperature of 50 °C, the recombinant protease exhibited the highest activity towards the substrate casein, though the highest stability was at lower temperatures and pH between 7.0 and 9.0. This study provided the direct evidence that extracellular proteases secreted by the antagonist A. pullulans PL5 played a role in the biocontrol activities against some postharvest pathogens of apple and peach.     

Effect of chitosan/nano-silica coating on the physicochemical characteristics of longan fruit under ambient temperature

A novel chitosan/nano-silica hybrid film was prepared using tetraethoxysilane as precursor by in situ sol–gel process, and characterized by transmission electron microscopy. Its effect on preservation quality of longan fruits (Dimocarpus longan Lour. cv Shijia) was investigated under ambient temperature. The present study revealed that the excellent semi-permeable film of chitosan/nano-silica markedly extended shelf life, reduced browning index, retarded weight loss and inhibited the increase of malondialdehyde amount and polyphenoloxidase activity in fresh longan fruit. In addition, the peroxidase activity of longan fruit coated with hybrid film was lower than that in other treatment fruits. Decreases in the contents of total soluble solids, titratable acidity and ascorbic acid were also significantly inhibited by hybrid films. These data indicated that the chitosan/nano-silica coating might provide an attractive alternative to improve preservation quality of fresh longan fruits during extended storage.     

Review: Utilization of antagonistic yeasts to manage postharvest fungal diseases of fruit

Significant losses in harvested fruit can be directly attributable to decay fungi. Some of these pathogenic fungi are also the source of mycotoxins that are harmful to humans. Biological control of postharvest decay of fruits, vegetables and grains using antagonistic yeasts has been explored as one of several promising alternatives to chemical fungicides, the use of which is facing increasingly more stringent regulation. Yeast species have been isolated over the past two decades from a variety of sources, including fruit surfaces, the phyllosphere, soil and sea water, and their potential as postharvest biocontrol agents has been investigated. Several mechanisms have been proposed as responsible for their antagonistic activity, including competition for nutrients and space, parasitism of the pathogen, secretion of antifungal compounds, induction of host resistance, biofilm formation, and most recently, the involvement of reactive oxygen species (ROS) in defense response. It has been recognized that a biocontrol system is composed of a three-way interaction between the host (commodity), the pathogen and the yeast, all of which are affected by environmental factors. Efficacy and consistent performance in controlling postharvest diseases are the hurdles that must be overcome if the use of yeast biocontrol agents and other alternatives are to be widely used commercially. Therefore, attempts have been made to combine alternative treatments in order improve their overall performance. The current review provides a brief overview of the topic of the use of yeasts as postharvest biocontrol agents and includes information on the sources from which yeast antagonists have been isolated, their mode of action, and abiotic stress resistance in yeast as it relates to biocontrol performance. Areas in need of future research are also highlighted.     

Novel implementation of laser ablation tomography as an alternative technique to assess grain quality and internal insect development in stored products

In the last century, there have been important advances in peering inside the internal grain environment. This has included electrical conductance mills, acoustical tools, near-infrared spectroscopy, and soft X-ray imaging. Each technique has their own advantages and limitations. In this current study, we describe a novel process for assessing grain quality and internal insect development in grain using laser ablation tomography (LATscan) and compare it to soft X-ray imaging for three life stages (larva, pupa, adult) of Sitophilus zeamais and Rhyzopertha dominica. Our results suggest that X-rays overestimated grain volume loss by about a quarter compared to LATscan. Moreover, LATscan produces a rich digital dataset on the level of microns that can be explored to ask biologically-relevant questions. We discuss the current limitations of LATscan in context with other techniques. Ultimately, the results highlight LATscan as a useful additional tool to further our understanding pest-commodity interactions in the postharvest supply chain.     

Comparison of efficacy of nitric oxide fumigation under nitrogen and carbon dioxide atmospheres in controlling granary weevil (Sitophilus granaries) and confused flour beetle (Tribolium confusum)

Nitric oxide (NO) was a recently discovered fumigant for postharvest pest control. Because NO reacts with oxygen (O₂) spontaneously to form nitrogen dioxide (NO₂), NO fumigation must be conducted under ultralow oxygen (ULO) atmospheres to preserve NO and nitrogen (N₂) has been used to establish ULO atmospheres in NO fumigation studies in the past. However, carbon dioxide (CO₂) can also be used to ULO atmospheres and CO₂ fumigation was also reported to be effective in controlling certain insect pests and enhancing toxicity of some fumigants. In the present study, NO fumigations under ULO conditions established with N₂ and CO₂ were compared for effects against granary weevil, Sitophilus granaries, and confused flour beetle, Tribolium confusum. All life stages of the two insects were subjected to 12 h fumigation treatments with 0.5–1.0% NO at 25 °C under ULO established with CO₂ (NO–CO₂ treatments) and N₂ (NO–N₂ treatments). The most tolerant life stage for each species in each fumigation treatment was then fumigated with NO for 24 h fumigation at 25 °C to determine an effective treatment. There were no significant differences in mortalities of adults, larvae, and pupae at all NO concentrations between ULO conditions established with CO₂ and N₂ for either insect. NO–CO₂ was, however, significantly less effective than NO–N₂ against eggs of both species. Granary weevil pupae and confused flour beetle eggs were the most tolerant stages for each respective species to NO fumigation: >99% mortality of granary weevil pupae and confused flour beetle eggs were, however, achieved in the 24 h fumigation with 2% and 1% NO, respectively. This study showed that CO₂ can be used to establish ULO atmospheres for NO fumigation, but did not significantly contribute to insect mortality.     

Effect of chemical and physical properties of Dried Distillers Grains with solubles (DDGS) on Tribolium castaneum (Herbst) development

Dried Distillers Grains with Solubles (DDGS) has been used as a grain-based supplement in animal feed. The focus of this study was to investigate the effects of chemical and physical characteristics that may affect DDGS susceptibility to Tribolium castaneum (Herbst) infestation. Multiple regression-based models were built to determine if the diet’s chemical and physical characteristics affect T. castaneum development. Based on the regression models, diet particle size was the most influential factor in larval development. Larval weight increased more rapidly on T. castaneum normal laboratory diet, 90% flour/10% brewer’s yeast (F/Y), followed by ground DDGS, compared to raw DDGS with different particle sizes. To determine the effect of diet particle size on larval development, experiments were conducted to increase particle size by pelletizing F/Y using a rotary drum granulation apparatus. T. castaneum larval stage lasted significantly longer on F/Y granules with a particle size of 1.4 mm or larger. Although the chemical properties of DDGS (protein, fat, fiber and ash content) were not significant predictors of T. castaneum larval development, larval developmental time and weight gain on ground DDGS was significantly slower than on F/Y, highlighting the potential importance of nutritional factors not accounted for in our analysis. Our results conclude that storing DDGS as raw ingredient and in the granule or pellet form with large particle size may help limit T. castaneum infestations. Although granulation or pelletization may not be economically feasible for small manufacturing plants, larger producers may benefit from the reduced risk of insect contamination associated with secondary feeders such as T. castaneum.     

Chitosan coatings enriched with essential oils: Effects on fungi involved in fruit decay and mechanisms of action

BackgroundPesticides have a negative impact on the health of consumers and on the environment, and as a consequence, the use of naturally occurring antimicrobial agents have become more popular to prevent food spoilage. An alternative is the use of chitosan, because of its antimicrobial, antioxidant activities and its capacity to form films having good mechanical properties. Chitosan has been used in the preparation of films and coatings for the preservation of different food products, and the addition of essential oils have been shown to be a good strategy to improve its antimicrobial activity in situ.Scope and approachThis review compiles information related to studies and research that employ chitosan as matrix for films, coatings and nanogels with essential oils extracted from plants, as antifungal agent. A special emphasis has been addressed to the effect exerted on the most pathogenic fungi for crops, also highlighting the mechanisms of action that researchers attribute to the composite, together with inhibition data.Key Findings and ConclusionEffective natural treatments, using chitosan-essential oils films or nano-emulsions, improve the preservation of fruit in terms of fungal decay. The effect is particularly dependent on the ability to release the antimicrobial compounds from the polymer matrix, the type of essential oils, the fungal species, and the incubation temperature. Several efforts are still required to understand in detail the mechanism of degradation of perishable food, how the chitosan-essential oils composites control or inhibit food decay, the effects on other pathogenic and non-pathogenic moulds as well as on the production of mycotoxins.     

Hermetic storage with plastic sealing to reduce insect infestation and secure paddy seed quality: A powerful strategy for rice farmers in Mozambique

Rice is the world's most important staple food and the basis of the diet of the majority of the population. In small farm agriculture, the yields obtained in cereal production are usually low and losses, both in the field and during storage, are dramatically high, particularly in developing countries. In Mozambique, these aspects, together with an increased frequency of floods, are hindering advances in rice production. Aimed at contributing to the reduction of losses in stored rice, trials were carried out to compare the effectiveness of traditional raffia bags and of hermetic storage using single and double plastic bags concerning quantitative losses and seed quality, including germination potential, after three and six months of storage. Pest identification, insect populations estimates, percentage of weight loss, germination power and seedling vigor were evaluated. The results showed that, in descending order of density, Angoumois grain moth (Sitotroga cerealella Olivier), lesser grain borer (Rhyzopertha dominica F.), rice/maize weevil (Sitophilus zeamais Mostch. and Sitophilus oryzae L.) and red flour beetle [Tribolium castaneum (Herbst)] were the main insects infesting the rice. When compared to hermetic storage with both single and duplicate airtight bags, traditional storage presented statistically significant higher mean infestation density (30.63–53.94 individuals/kg in traditional and 0.71–3.50 individuals/kg in hermetic storage) and percentage of weight loss (3.03–3.44% in traditional contrasting with 0.27–0.47% in hermetic conditions). In traditional storage a significant 38.25% drop in the germination potential was also observed, attaining values below the established minimum tolerated in Mozambique (80%), while under hermetic storage, that reduction remained within the acceptable values of 13.9–17.5%. The distinct storage methods did not produce significant differences on the moisture content of the grain. These results demonstrate that the use of hermetic storage has resulted in a safe, pesticide-free, and sustainable storage method, suitable for rice seeds, with advantages over traditional bagging. The results presented here lead to propose hermetic storage to be adopted by paddy small farmers, in order to lever food security and income generation in the country.     

Short anoxic treatments to enhance <Emphasis Type="Italic">trans</Emphasis>-resveratrol content in grapes and wine

In a search for a method to improve the (endogenous) trans-resveratrol content in post harvested grapes, several short anoxic treatments with dry nitrogen have been assayed. A significant enhancement of the trans-resveratrol content was obtained, especially for treatments under 24 h; moreover, with shorter treatments (e.g. 6 or 15 h) the induced higher content of trans-resveratrol was maintained during several days with no appreciable anoxic damage of the grapes. These results allowed the design of an anoxic treatment protocol for wine grapes prior to the vinification process that led to a trans-resveratrol enriched wine.     

A comparison of integrated pest management and traditional practices for bagged maize stored in Nigeria

In grain markets in Nigeria, stored product insects can cause significant losses in quality and quantity of stored grains. Insect infestations in storehouses are usually controlled by the unilateral use of chemical insecticides, which have unintended adverse effects. In this study, approximately 15 MT of well-dried, Aflasafe™-treated maize was procured from a local farm settlement in January 2016. It was subsequently fumigated, mechanically cleaned and placed in 100-kg polypropylene bags for storage. The study was conducted during February–December 2016, to compare the effectiveness of traditional storage practice (TSP) with integrated pest management (IPM) practice. IPM for warehouse facilities focuses on adequate drying, inspection and cleaning of storehouses and their surroundings and regular examination of grain (IPMD). Traditional storage practice generally has little or no sanitation of the facility or regular examination of grain (monitoring) for insect pests. Study locations were markets in Ibadan, Oyo and Ilorin towns in Southwest and Northcentral Nigeria, respectively. Each market had two storehouses where TSP or IPMD was assigned; each storehouse had twenty-five bags of maize, which were sampled monthly to assess the effects of the two practices on stored maize insect populations and quality. Generally, from October to December, there tended to be more insects of all species in TSP than IPMD. Percent insect damaged kernels (%IDKNB) and weight loss (%WL) were also higher in TSP than IPMD during the October–December period. Percent germination in December was higher in IPMD (96.3%) than in TSP (85.3%). Aflatoxin levels in both TSP and IPMD did not exceed 1.9 ppb. Data from this study show that IPMD results in lower stored-product insect population levels and better maize quality than TSP. Thus, IPMD practice needs to be more widely adopted in storehouses in Nigeria and the rest of sub-Saharan Africa.