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Botrytis cinerea causes postharvest fruit rot of an infinity of crops, the infective capacity is due to its physiological diversity that shown, even inside the same crop. For its control, the use of antagonistic microoganisms is emerging as a sustainable option. In the present work, 40 Botrytis isolates from three vineyards were characterized by their ability to infect grape fruit (Thomson Seedless), the results showed that all produced lesions diameters from 6.5 to 22.2 mm. Ten of these isolates that presented differences in terms of their virulence, were subject to in vitro antagonism test, using the yeasts Metschnikowia sp. NB9 and FLL17 (Kodamaea sp. FLL17 and the bacteria FR4B12 Bacillus sp. R4B12 from must and flower and fruit, respectively. The results showed that, on average, FRB412 had the highest inhibitory activity on the growth of Botrytis strains, exhibiting mycelial growth inhibition percentages from 51 to 81 %, followed by FLL17 (21 to 53 %) and NB9 (15 to 51 %). In conclusion, the three study strains have different ranges of biocontrol on Botrytis, whose application could reduce gray rot in grapes.

A pre-adaptation approach to osmotic stress was used to improve the colonisation and biocontrol capacity of Yarrowia lipolytica M18/3b on the apple under pre- and postharvest conditions. For this, ...

Background Two edible coating (EC) emulsions based on potato starch (F6 and F10) alone or formulated with sodium benzoate (SB, 2% w/w) (F6/SB and F10/SB) were evaluated to maintain postharvest quality of cold-stored 'Fino' lemons and control sour rot on lemons artificially inoculated with Geotrichum citri-aurantii. Previous research showed the potential of these ECs to improve the storability of 'Orri' mandarins and reduce citrus green and blue molds caused by Penicillum digitatum and Penicillium italicum, respectively. Results The coatings F6/SB and F10/SB significantly reduced sour rot incidence and severity compared to uncoated control samples on lemons incubated at 28 °C for 4 and 7 days. The F6/SB coating reduced weight loss and gas exchange compared to uncoated fruit after 2 and 4 weeks of storage at 12 °C plus a shelf life of 1 week at 20 °C, without adversely affecting the lemon physicochemical quality. Conclusion Overall, the F6/SB coating formulation, composed of pregelatinized potato starch, glyceryl monostearate, glycerol, emulsifiers and SB, with a total solid content of 5.5%, showed the best results in reducing citrus sour rot and maintaining the postharvest quality of cold-stored 'Fino' lemons. Therefore, it showed potential as a new cost-effective postharvest treatment suitable to be included in integrated disease management programs for citrus international markets with zero tolerance to chemical residues. © 2021 Society of Chemical Industry.

Starch is a biodegradable polymeric carbohydrate that can easily form films and coatings and can readily be obtained from some food industry by-products and wastes, which may contribute to the circular bioeconomy. In this work, we studied the potential of two edible coating emulsions based on pregelatinized potato starch (PPS) and glyceryl monostearate (GMS) alone (F6 and F10) or formulated with the food additive sodium benzoate (SB, 2%) (F6/SB and F10/SB) to control sour rot, an important citrus postharvest disease caused by the fungus Geotrichum citri-aurantii, and maintain postharvest quality of cold-stored ‘Orri’ mandarins. The PPS-GMS coating application was compared to dipping in water (uncoated controls) and dipping in a 2% SB (w/v) aqueous solution. The results showed that the coating F10/SB was the most promising treatment to control sour rot on mandarins, with reductions in disease incidence with respect to the uncoated control samples of 94, 69, and 55% after 2, 4, and 6 weeks of storage at 5 °C, respectively. Coatings formulated without SB were ineffective. Regarding fruit quality, the coating F10 was the most effective to reduce weight loss, maintain firmness, and provide gloss on mandarins stored at 5 °C for up to 6 weeks followed by a shelf-life period of 1 week at 20 °C. The addition of SB to the PPS-GMS coatings adversely affected these coating properties, but the coating F10/SB still reduced weight loss compared to uncoated controls without negatively affecting the fruit physicochemical (juice titratable acidity, soluble solids content, and volatiles content) and sensory quality (overall flavor, off-flavors, external aspect). Overall, the coating F10/SB showed the greatest potential for commercial use as an efficient and environmentally friendly alternative to conventional fungicides and waxes for sour rot control and quality preservation of cold-stored mandarins.

The low quality of the apple produced in Queretaro, Mexico, impedes its efficient fresh marketing. One possible solution to this problem is to produce champagne-type sparkling cider. Therefore, the objective of this work was to select strains of Saccharomyces yeasts isolated from apple varieties of the region, based on desirable traits to make sparkling ciders. To select the yeasts, we induced the spontaneous fermentation of the must of 14 varieties, from which 135 strains (102 Saccharomyces spp.) were isolated and three (MM7, 436.4 and RY5) were selected, all with killer phenotype, β-glucosidase activity, tolerance to 8 % ethanol and 50 mg∙L-1 of SO2, a different degree of flocculation and with the ability to produce more than 3 atm of pressure in the bottle. With MM7, a base cider of good quality was obtained (glucose < 2 g∙L-1, pH 3.57, total titratable acidity of 4.03 g∙L-1 and volatile acidity of 0.27 g∙L-1). For the second fermentation, the base ciders made with MM7 were bottled, 15 g∙L-1 of sucrose were added and then they were inoculated separately with MM7, 436.4, RY5 or K1-V1116. By day 21, they all reached about 6 atm of pressure. Sensorially, ciders made with MM7 stood out in effervescence speed, and those made with RY5 in taste acceptability. The amplification and sequencing of the ITS1/ITS4 domain identified MM7 and 436.4 as S. cerevisiae, and RY5 as S. paradoxus. The selected yeasts showed desirable traits for the production of quality sparkling ciders typical of the region.

Antifungal composite edible coatings (ECs) formulated with pregelatinized potato starch (PPS, 1.0-2.0 % w/w) as biopolymer, glyceryl monostearate (GMS, 0.5-1.5 %, w/w) as hydrophobe, glycerol (Gly, 0.5-1.5 %, w/w) as plasticizer, and sodium benzoate (SB, 2 % w/w) as antifungal agent were optimized using the Box–Behnken response surface methodology to extend the postharvest life of Orri’ mandarins. The second order polynomial models satisfactorily fitted the experimental data, with high values of the coefficient of determination for the different variables (R2>0.91). The individual linear effect of GMS concentration was significant in all the responses evaluated, whereas PPS only affected emulsion viscosity, fruit tacking, and weight loss of coated mandarins. Gly only affected acetaldehyde content in the juice of coated mandarins when interacted with PPS and in the quadratic effect. The optimum concentrations of PPS, GMS, and Gly for the starch-based EC based on maximum fruit quality and required emulsion properties were predicted to be 2.0, 0.5 and 1.0 % (w/w), respectively. The optimal EC reduced weight loss of mandarins and created a modified atmosphere within the fruit without negatively affecting the overall acceptability of the fruit. On the other hand, the optimized antifungal EC containing SB significantly reduced postharvest green and blue molds and sour rot on mandarins artificially inoculated with the pathogens Penicillium digitatum, Penicillium italicum and Geotrichum citri-aurantii, respectively. Therefore, the optimized antifungal EC showed potential to control the main postharvest diseases and maintain the overall quality of ‘Orri’ mandarins and could be a suitable alternative to commercial citrus waxes formulated with conventional chemical fungicides.

Pantoea agglomerans strain CPA-2 is an effective biocontrol agent (BCA) for postharvest diseases of citrus and pome fruits. However, for registration purposes and to implement their use as effective control strategy, it is necessary to study the traceability and survival of BCAs in their target application sites. The main objective of this work was to evaluate the persistence and quantify the population of CPA-2 after its postharvest application on orange cultivar 'Valencia Late' by molecular techniques. After application, the persistence of CPA-2 was evaluated by sampling the packing line and storage chambers, as well as on clothing of the workers by conventional PCR. The results showed that the maximum persistence of CPA-2 was lower than 3 days in surfaces of packing line. Furthermore, CPA-2 did not survive more than 1 day on working clothes, while in the environment or on different storage chamber surfaces it was not detected. In addition, the CPA-2 populations were quantified by quantitative PCR (qPCR) combined with a DNA intercalating reagent, propidium monoazide dye (qPCR-PMA) to quantify the CPA-2 viable cells on fruit surface. The qPCR-PMA method was compared with qPCR and dilution plating method. Results showed that CPA-2 populations quantified by qPCR-PMA were significantly different compared with those obtained by qPCR during the time-course of the assay; however, no significant differences were observed between qPCR-PMA and dilution plating. In conclusion, the persistence of CPA-2 was low at different sampling areas, suggesting that it cannot grow and survive on the surfaced sampled. Furthermore, qPCR-PMA method can be a quick and specific tool to monitor the viable population of CPA-2 on fruit surface.

Pantoea agglomerans strain CPA-2 is an effective biocontrol agent (BCA) against the major postharvest pathogens present on pome and citrus fruits. Dehydration, such as freeze-drying, spray-drying and fluidized bed drying is one of the best ways to formulate BCAs. In this work, the survival of CPA-2 cells after formulation was determined by dilution plating and molecular methods as qPCR alone and combined with a sample pretreatment with a propidium monoazide dye (PMA-qPCR) and they were used to calculate treatment concentrations in efficacy trials on postharvest oranges. Furthermore, no significant differences in CPA-2 survival were observed as determined by dilution plating and PMA-qPCR after both the freeze drying and fluidized bed drying processes; however, an interesting significant difference was observed in the spray dried product comparing all quantitative methods. A difference of 0.48 and 2.17 log10 CFU or cells g/dw was observed among PMA-qPCR with qPCR and dilution plating, respectively. According to our study, dilution plating was shown to be an unreliable tool for monitoring the survival of CPA-2 after spray drying. In contrast, the combination of PMA and qPCR enabled a quick and unequivocal methodology to enumerate viable and VBNC CPA-2 cells under stress-dried conditions. Efficacy trials showed that, after 3 days, spray drying formulation rehydrated with 10% non-fat skimmed milk (NFSM) was as effective as fresh cells to control Penicillium digitatum in oranges.

The aim of this study was to evaluate the environmental fate and behaviour of the biocontrol product based on Pantoea agglomerans CPA-2 cells after its release into the field. Three different methods, dilution plating, quantitative real-time PCR (qPCR) and propidium monoazide combined with qPCR (PMA-qPCR) were used to track the CPA-2 population. Populations quantified by qPCR were statistically different compared with those by the PMA-qPCR and dilution plating methods. In addition, the spray dispersion of the CPA-2 treatment was evaluated using water-sensitive papers. A lack of dispersion of the CPA-2 treatment was observed at distances of 2.5 ± 0.5 m. The presence and persistence of CPA-2 in the environment were monitored in different areas, by dilution plating and confirmed by conventional PCR. The results showed that CPA-2 can survive but not proliferate on weed and leaves. The persistence on inert surfaces, such as motorised backpack sprayer, gloves and working clothes, was less than 7 days. In conclusion, PMA-qPCR was a potential tool for fast and specific monitoring of viable populations of CPA-2 and it gave valuable information on population behaviour. Moreover, the results demonstrate that CPA-2 would not present a risk for the environment because of its low establishment, survival and dispersion.

Pantoea agglomerans strain CPA-2 is an effective biocontrol agent (BCA) for postharvest diseases of citrus and pome fruit. To implement their use as a control strategy is necessary to study the traceability of BCAs in the environment during application, for registration issues. In this study, the presence and persistence of CPA-2 was monitored in the packing line, storage chambers and on working clothes by conventional PCR. After postharvest application, the presence of CPA-2 was not detectable in the environment and storage chambers, whereas on working clothes and the packing line its persistence was less than 1 and 3 days, respectively. Additionally, the CPA-2 population was quantified on oranges stored at two different temperatures (20 °C and 4 °C) by quantitative PCR (qPCR), sample pretreatment with a propidium monizade dye (PMA–qPCR) and the dilution plating method. At the initial time of the assay, no differences were observed in CPA-2 populations quantified by qPCR, PMA–qPCR, and dilution plating, at both storage temperatures. However, CPA-2 populations quantified by PMA–qPCR were significantly different compared with those obtained by qPCR during the time-course of the assay; no significant differences were observed between PMA–qPCR and dilution plating. In conclusion, the persistence of P. agglomerans CPA-2 at different sampling areas after postharvest application was low. Furthermore, PMA–qPCR gave valuable information on viable population behavior and the presence of residual DNA from dead cells. In general, these studies help to understand the persistence of antagonists when applied under postharvest conditions and will lead to optimization of time and mode of application.

The aim of this study was to determine the effects of maturity stage, ripening time, year, and several quality parameters of the fruit (total soluble solids, acidity, pH, firmness, and color) on the susceptibility to Penicillium expansum of apple genotypes established in Queretaro, Mexico. The accessions consisted in cultivars, creoles and low chilling hybrids obtained in Mexico. The development of the disease was determined by measuring the lesion diameter of decay (severity) on fruits inoculated with 104 spores mL−1 and incubated for 8 days at 26 °C. Data were analyzed for significant difference and for significant correlation by analysis of variance and principal component analysis (PCA), respectively. In 2011, commercial maturity apples exhibited a greater tolerance to P. expansum than did over-mature apples (lesion diameters of 31.3 and 34.9 mm, respectively); however, the ripening time of genotypes did not influence the development of the pathogen in either year of study (21.8, 23.2, and 25.5 mm in diameter for early-, middle-, and late-ripening genotypes, respectively, in 2012). Differences in the susceptibility of genotypes were observed in both harvest years; the most outstanding were ‘436’, ‘442-9’ and ‘441’ (17.4, 17.5 and 17.6 mm, respectively) in contrast to ‘Golden Delicious’ (34.2 mm) in 2012; nevertheless, no correlation was observed in genotypes behavior between the two harvest years (r = 0.11NS). Excluding total soluble solids in 2012, PCA analysis did not reveal any correlation between pathogen severity and fruit quality parameters, suggesting the possibility that other mechanisms are involved in the fruit-pathogen interaction. This study demonstrated for the first time that ripening time does not affect the susceptibility to P. expansum in apple fruits.

Dilution plating is the quantification method commonly used to estimate the population level of postharvest biocontrol agents, but this method does not permit a distinction among introduced and indigenous strains. Recently, molecular techniques based on DNA amplification such as quantitative real-time PCR (qPCR) have been successfully applied for their high strain-specific detection level. However, the ability of qPCR to distinguish viable and nonviable cells is limited. A promising strategy to avoid this issue relies on the use of nucleic acid intercalating dyes, such as propidium monoazide (PMA), as a sample pretreatment prior to the qPCR. The objective of this study was to optimize a protocol based on PMA pre-treatment samples combined with qPCR to distinguish and quantify viable cells of the biocontrol agent P. agglomerans CPA-2 applied as a postharvest treatment on orange. The efficiency of PMA-qPCR method under the established conditions (30μM PMA for 20min of incubation followed by 30min of LED light exposure) was evaluated on an orange matrix. Results showed no difference in CFU or cells counts of viable cells between PMA-qPCR and dilution plating. Samples of orange matrix inoculated with a mixture of viable/dead cells showed 5.59log10 CFU/ml by dilution plating, 8.25log10 cells/ml by qPCR, and 5.93log10 cells/ml by PMA-qPCR. Furthermore, samples inoculated with heat-killed cells were not detected by dilution plating and PMA-qPCR, while by qPCR was of 8.16log10 cells/ml. The difference in quantification cycles (Cq) among qPCR and PMA-qPCR was approximately 16cycles, which means a reduction of 65,536 fold of the dead cells detected. In conclusion, PMA-qPCR method is a suitable tool for quantify viable CPA-2 cells, which could be useful to estimate the ability of this antagonist to colonize the orange surface.

The registration of biological control agents requires the development of monitoring systems to detect and quantify the agent in the environment. Pantoea agglomerans CPA-2 is an effective biocontrol agent for postharvest diseases of citrus and pome fruits. The monitoring of CPA-2 in postharvest semi-commercial trials was evaluated by Rodac impression plates and the colonies isolated were confirmed by conventional PCR using the SCAR primers PAGA1 and PAGB1. Samples were taken from different surfaces that had contact with CPA-2, the surrounding environment and working clothes worn by handlers. Moreover, population dynamics of the strain CPA-2 were determined on apple surfaces using both the classical plating technique and real-time quantitative PCR (qPCR). A qPCR assay using a 3'-minor groove-binding (MGB) probe was developed for the specific detection and quantification of P. agglomerans strain CPA-2. Based on the nucleotide sequence of a SCAR fragment of CPA-2, one primer set and TaqMan MGB probe were designed. The primers SP2-F/SP2-R and the TaqMan MGB probe showed a specific detection of strain CPA-2 on apple surfaces, which was verified tested against purified DNA from 17 strains of P. agglomerans, 4 related Pantoea species, and 21 bacterial strains from other genera isolated from whole and also freshly-cut fruit and vegetables. The detection level was approximately 10(3) cells per reaction, and the standard curve was linear within a range of 5log units. Results from semi-commercial trials showed that CPA-2 had a low impact. The maximum persistence of P. agglomerans CPA-2 was not longer than 5days in plastic boxes stored at 0°C. Significant differences in CPA-2 population level dynamics were observed in results obtained by qPCR and dilution plating. These differences may indicate the presence of non-degraded DNA from non-viable cells. In conclusion, qPCR is a novel potential tool to quickly and specifically monitor recent surface colonisation by CPA-2 populations on apple surfaces during large-scale experiments that could ensure efficient and successful treatments.

Native lactic acid bacteria (LAB) are capable of growing during winemaking, thereby strongly affecting wine quality. The species of LAB present in musts, wines during malolactic fermentation (MLF), and barrels/filters were investigated in wineries from the emerging wine region of Queretaro, México using multiplex PCR and culture. The resistance to wine-like conditions (WLC): ethanol (10, 12, and 13%), SO2 (30 mg⋅l-1), and low pH (3.5) of native LAB strains was also studied. Five species were detected within 61 samples obtained: Oenococcus oeni, Lactobacillus plantarum, Pediococcus parvulus, Lactobacillus hilgardi, and Lactobacillus brevis. Four species (excepting L. brevis) were found in must; O. oeni and P. parvulus were ubiquitous in wine and L. plantarum and L. brevis were mainly present at the initial stage of MLF, while L. hilgardii was mostly detected at the advanced stage. Furthermore, some species detected in barrel/filter, prove them to be hazardous reservoirs. From 822 LAB isolates, only 119 resisted WLC with 10% ethanol; the number of strains able to grow in WLC with 13% ethanol decreased approximately by 50%, O. oeni being the most versatile species with 65% of resistant isolates, while Lactobacillus spp. and P. parvulus were the most strongly affected, especially those recovered from barrel/filter, with less than 10% of resistant isolates. This study evidences the presence of local strains able to be used as starter cultures, and also enabled the assessment of the risks derived from the presence of spoilage LAB strains resistant to WLC.

La producción de frutas y hortalizas bajo invernadero se ha incrementado en México de 721 ha en 1996 a 9,500 ha en 2009. Recientemente se presentó en el pimiento (Capsicum annum L.) una enfermedad fungosa que afecta al pedúnculo del fruto en poscosecha y cuyo agente causal es Fusarium stilboides. Una alternativa para reducir el daño producido por este hongo es el uso de métodos de control biológico. Para evaluar el poder antagónico de cepas de levaduras de distinto origen en comparación con métodos químicos para el control de F. stilboides, se inocularon frutos de pimiento con una concentración conocida de levaduras (107 UFC·mL-1), en presencia de una suspensión de esporas (105 UFC·mL-1) de F. stiboides, o bien se sumergieron en soluciones de distintos fungicidas (benomyl, captán, bicarbonato de sodio y bicarbonato de potasio) a diversas concentraciones y se inocularon con la suspensión de esporas del hongo. Benomyl 0.6 g·L-1 mostró el más alto nivel de inhibición de la severidad en el pedúnculo (81 %), mientras que bicarbonato de sodio 30 g·L-1 resultó más efectivo a nivel del epicarpio (51 % de reducción del diámetro de la lesión). Las levaduras 22-2 5b y 18-1 x de manzana, Avv, 5vtt (Candida incommunis) de velos de vino, 3d. RMA3 de pimiento redujeron el diámetro de la lesión en el epicarpio en 63, 54, 53, 47 y 45 %, respectivamente. Greenhouse production of fruits and vegetables in Mexico has increased from 721 ha in 1996 to 9,500 ha in 2009. A fungal disease caused by Fusarium stilboides has recently appeared in pepper (Capsicum annum L.) that affects the fruit peduncle in the postharvest period. An alternative means of reducing the damage caused by this fungus is the use of biological control methods. To assess the antagonistic capacity of yeast strains of different origin to control F. stilboides and compare them with chemical methods, pepper fruits were inoculated with a known yeast suspension (107 CFU•mL-1) and a spore suspension of F. stilboides (105 CFU•mL-1), or they were immersed in different fungicidal solutions (benomyl, captan, sodium bicarbonate and potassium bicarbonate) at various concentrations and then inoculated with the fungal spore suspension. Benomyl at 0.6 g.L-1 showed the highest severity-inhibition level on the peduncle (81 %), while sodium bicarbonate at 30 g.L-1 was the most effective on the epicarp (51 % reduction in lesion diameter). Yeasts-25b and 18-1x from apple, Avv, 5vtt (Candida incommunis) from wine veils and 3d. RMA3 from pepper reduced lesion diameter on the epicarp by 63, 54, 53, 47 and 45 % respectively.