In vitro and in vivo antimicrobial activity of Xenorhabdus bovienii YL002 against Phytophthora capsici and Botrytis cinerea

Aims: Developing new bio‐agents to control plant disease is desirable. Entomopathogenic bacteria Xenorhabdus spp. have potential antimicrobial activity in agriculture. This work was conducted to evaluate the antimicrobial activity of Xenorhabdus bovienii YL002 on plant pathogenic fungi and oomycete in vitro and the efficiency of this strain to reduce the in vivo incidence of grey mould rot on tomato plants caused by Botrytis cinerea and leaf scorch on pepper plants caused by Phytophthora capsici. Methods and Results: The antimicrobial activity of X. bovienii YL002 was firstly determined on in vitro plant pathogenic fungi and oomycete and then on tomato fruits and plants infected with B. cinerea and pepper plants infected with P. capsici. The cell‐free filtrate of X. bovienii YL002 exhibited highest inhibition effects (>98%) on mycelia growth of P. capsici and B. cinerea. The 50% inhibition concentration (EC₅₀) of the methanol‐extracted bioactive compounds (methanol extract) of the cell‐free filtrate against P. capsici and B. cinerea were 164·83 and 42·16 μg ml^?1. The methanol extract also had a strong effect on the spore germination of P. capsici and B. cinerea, with a EC₅₀ of 70·38 and 69·33 μg ml^?1, respectively. At 1000 μg ml^?1, the methanol extract showed a therapeutic effect of 70·82% and a protective effect of 77·4% against B. cinerea on tomato plants compared with the control. The methanol extract also showed potent effect against P. capsici, with a therapeutic effect of 68·14% and a protective effect of 65·46% on pepper plants compared with the control. Conclusions: Xenorhabdus bovienii YL002 produces antimicrobial compounds with strong activity on plant pathogenic fungi and oomycete and has the potential for controlling grey mould rot of tomato plants and leaf scorch of pepper and could be useful in integrated control against diverse plant pathogenic fungi and oomycete. Significance and Impact of the Study: This study showed the potential that X. bovienii YL002 can be used to control the grey mould rot caused by B. cinerea on tomato plants and leaf scorch caused by P. capsici on pepper plants with the objective to reduce treatments with chemical fungicides.     

Distribution ofGeotrichum candidum citrus race in citrus groves and non-citrus fields in Japan

The presence ofGeotrichum candidum citrus race, the citrus sour rot pathogen, was examined in soils of citrus groves and non-citrus fields of Japan. Soil samples were collected from 223 sites (118 sites in citrus groves, and 105 sites in fields cultivated with 33 species of non-citrus plants and in evergreen broad-leaved forest) in 11 main citrus-growing prefectures, and Hokkaido, a non-citrus-growing area. Of 236 soil samples from citrus groves, 95.76% containedG. candidum citrus race and 0.42% contained the non-citrus race; while of 210 samples from non-citrus fields, 62.85% and 4.76% contained the citrus race and the non-citrus race respectively. All of the citrus race isolates obtained either from citrus groves or non-citrus fields were pathogenic on lemon (Citrus limon) and satsuma mandarin (Citrus unshiu), but some of these isolates failed to infect orange (Citrus sinensis). The non-citrus races were pathogenic on ripe tomato fruit (Lycopersicon esculentum) and ripe muskmelon fruit (Cucumis melo var.reticulatus). Results indicated that citrus sour rot pathogen is widely distributed in citrus groves and non-citrus fields of diverse plant species in Japan.     

Effect of various salts on the growth and development of Geotrichum candidum,the causal agent of carrot sour rot

This study evaluated the efficacy of ammonium, calcium, potassium and sodium salts as possible alternatives to synthetic fungicides in the control of Geotrichum candidum, the causal agent of sour rot on carrots. In vitro mycelial growth of G. candidum was completely halted by ammonium bicarbonate and carbonate; calcium oxide; potassium benzoate, carbonate and sorbate; sodium benzoate, carbonate and fluoride (2% w/v). Potassium and sodium bicarbonate also reduced mycelial growth by 77.78% and 90.60%, respectively, and the difference between the effects of sodium bicarbonate and the first group of salts was not statistically significant (p < 0.05). With the exception of potassium and sodium bicarbonate, the above‐mentioned salts also halted or strongly reduced arthrospore germination. Potassium bicarbonate, and sodium bicarbonate, acetate and propionate significantly increased conidiation (p < 0.05). Of all the salts tested in vitro, only ammonium bicarbonate and carbonate, calcium oxide and sodium fluoride were toxic to G. candidum. In in vivo studies, all the calcium salts tested (acetate, chloride, citrate, formate, lactate, oxide, propionate and silicate), several of the sodium salts (acetate, bicarbonate, chloride and fluoride) and potassium bicarbonate exhibited both protective and curative activity against G. candidum, significantly reducing the severity of sour rot in comparison to pathogen‐inoculated controls (p < 0.05). Although no curative was observed with ammonium bicarbonate, ammonium carbonate, potassium carbonate, potassium chloride, sodium carbonate or sodium citrate, these salts also demonstrated significant protective activity against sour rot when compared to controls (p < 0.05). In sum, the study findings show that all of the selected salts may be used to control carrot sour rot, except for sodium fluoride, which exhibited phytotoxicity to carrots.     

Parasitic specialization ofGeotrichum candidum citrus race

Parasitic specialization ofGeotrichum candidum citrus race, the causal agent of citrus sour rot, was investigated. Of seven isolates tested for pathogenecity, all could infect ten species of citrus fruits and edible parts of five species of noncitrus crops. Only one isolate (Ap2), isolated from soil of an apple orchard, could infect apple fruit.     

Effect of allelopathic compounds produced by Cistus ladanifer on germination of 20 Mediterranean taxa

In order to analyze the extent to which allelopathic action of Cistus ladanifer may influence the successional progression towards Mediterranean sub-climacic shrublands and the geographical distribution of other species, the inhibitory effect of Cistus ladanifer extracts on the germination of 20 Mediterranean species was analyzed. Five of the species tested were characteristic of maquis sub-climacic shrublands: Arbutus unedo, Adenocarpus argyrophyllus, Phillyrea angustifolia, Phillyrea latifolia, and Rhamnus alaternus. Tests were also carried out on 6 Cistaceae species in order to evaluate the auto-toxicity rate of allelopathic extracts: Halimium umbellatum subsp. viscosum, Halimium ocymoides, Cistus ladanifer, Cistus salvifolius, Cistus monspeliensis, and Cistus populifolius. Nine herbaceous species sharing the natural habitat with C. ladanifer were also examined. Results confirmed a clear inhibitory and delaying effect of aqueous C. ladanifer-leaf extracts on the germination of P. angustifolia, P. latifolia, R. alaternus, H. ocymoides, C. populifolius, Erysimum lagascae, Brassica barrelieri, Silene tridentata, and Moricandia moricandioides. Assays with soil collected below the canopy of the C. ladanifer community showed more pronounced inhibitory effect on sub-climacic shrub species than the aqueous extracts. In contrast, the opposite pattern was detected when analyzing the allelopathic effect of natural soil on Cistaceae and herbaceous species. Allelopathic compounds produced by C. ladanifer showed little auto-toxicity. The inhibitory effect of phytotoxic compounds accumulated in the jaral soil upon germination of A. unedo, B.␣barrelieri, and M. moricandioides was eliminated by heating soil at 150 °C for 10 min. Phenolic compounds (i.e. ferulic, p-hydroxybenzoic, vanillic, p-coumaric, and caffeic acids) and terpenes (i.e. α and β-pinene) with allelopathic documented effect were detected in the aqueous extracts and soil samples used in the analyses. We suggest that the allelopathic effect of C. ladanifer may influence the composition and structure of Mediterranean communities where the species is present, since it hinders the establishment of some sub-climacic species and may reduce the area occupied by numerous herbaceous species.     

Effects of volatile compounds on arthrospore germination and mycelial growth ofGeotrichum candidum citrus race

Three groups of volatile compounds, i.e., alcohols, aldehydes and esters, were tested for their effects on arthrospore germination and mycelial growth ofGeotrichum candidum citrus race, the causal agent of citrus sour rot. Alcohols (heptanol, octanol, nonanol, decanol, geraniol, citronellol) at a concentration of 1.0 μl/ml showed 60% or more inhibitory effects on both germination and mycelial growth of this pathogen. Among aldehydes tested, only citral had an inhibitory effect of more than 50%, while esters had no effect. The chain length of straight-chain (C6–C12) alcohols correlated with inhibitory effect, and nonanol (C9) was most active. Treatment with alcohols or citral prior to inoculation reduced colonization and maceration of lemon peel by this pathogen by 70% or more. Results suggested that alcohols or citral can probably be used to prevent the development of citrus sour rot.     

Sensitivity to boscalid in field isolates of Sclerotinia sclerotiorum from rapeseed in Henan Province,China

Sclerotinia stem rot caused by Sclerotinia sclerotiorum is an important disease of oilseed rape in Henan province of China. Boscalid belongs to succinate dehydrogenase inhibitor (SDHI) fungicides, many of which have strong antifungal activity against S. sclerotiorum. In 2015, a total of 175 isolates of S. sclerotiorum were collected from diseased oilseed rape plants in seven different regions of Henan Province. The EC₅₀ values of 175 isolates of S. sclerotiorum to boscalid ranged from 0.0073 to 0.3880 μg ml^?1, and the mean EC₅₀ value was 0.15 ± 0.09 μg ml^?1. The frequency distribution was unimodal. There was no cross‐resistance between boscalid and carbendazim, procymidone, iprodione, dimethachlone, fludioxonil or fluazinam. Field experiments showed that control efficacies of treatments with boscalid (50% WG) at 225, 300 and 375 g ai ha^?1 were 71%, 81% and 90%, respectively. In contrast, the control efficacy of carbendazim (50% WP) at 1,500 g ai ha^?1 was only 52%.     

Antimicrobial activities of therapeutic herbal plants against Listeria monocytogenes and the herbal plant cytotoxicity on Caco-2 cell

Aims: This study investigated the antimicrobial effect of various therapeutic herbal plants on Listeria monocytogenes, and their cytotoxicity effect on mammalian cells. Methods and Results: The extracts from 69 therapeutic herbal plants were used to investigate the effect on the growth inhibition of L. monocytogenes, and their minimal inhibition concentrations and minimal bactericidal concentrations were determined. Among the plants, Psoraleae semen L. (Bogolji) and Sophorae radix L. (Gosam) extracts, which showed obvious antilisterial activity, were examined for the stability to heat, NaCl and acidic condition. Moreover, cytotoxicities of Bogolji and Gosam were tested, using Caco‐2 cells. L. monocytogenes growth was completely inhibited by Bogolji and Gosam extracts at 3·2–6·3 and 50–100 AU ml^?1, respectively, and heat, NaCl and acidic condition did not affect the antilisterial activity of Bogolji and Gosam. Cytotoxic activities were observed only at high concentration (50 AU ml^?1) of Bogolji extract. Conclusion: Bogolji and Gosam could be considered as potential phytochemicals to control L. monocytogenes. Significance and Impact of the Study: Use of therapeutic herbal plants should be useful in controlling L. monocytogenes, because most consumers have better acceptance for phytochemicals than synthetic chemicals.     

Ascomycetous yeast species recovered from grapes damaged by honeydew and sour rot

Aims: To identify ascomycetous yeasts recovered from sound and damaged grapes by the presence of honeydew or sour rot. Methods and Results: In sound grapes, the mean yeast counts ranged from 3·20 ± 1·04 log CFU g^?1 to 5·87 ± 0·64 log CFU g^?1. In honeydew grapes, the mean counts ranged from 3·88 ± 0·80 log CFU g^?1 to 6·64 ± 0·77 log CFU g^?1. In sour rot grapes counts varied between 6·34 ± 1·03 and 7·68 ± 0·38 logCFU g^?1. Hanseniaspora uvarum was the most frequent species from sound samples. In both types of damage, the most frequent species were Candida vanderwaltii, H. uvarum and Zygoascus hellenicus. The latter species was recovered in high frequency because of the utilization of the selective medium DBDM (Dekkera/Brettanomyces differential medium). The scarce isolation frequency of the wine spoilage species Zygosaccharomyces bailii (in sour rotten grapes) and Zygosaccharomyces bisporus (in honeydew affected grapes) could only be demonstrated by the use of the selective medium ZDM (Zygosaccharomyces differential medium). Conclusions: The isolation of several species only from damaged grapes indicates that damage constituted the main factor determining yeast diversity. The utilization of selective media is required for eliciting the recovery of potentially wine spoilage species. Significance and Impact of the Study: The impact of damaged grapes in the yeast ecology of grapes has been underestimated.     

Antifungal activity of four weedy plant extracts against selected mycotoxigenic fungi

Aims: To investigate the antifungal activity of aqueous and organic extracts of four weedy plant species viz. Tagetes minuta, Lippia javanica, Amaranthus spinosus and Vigna unguiculata against isolates of four agriculturally important fungi, i.e. Fusarium verticillioides, F. proliferatum, Aspergillus flavus and A. parasiticus. Methods and Results: Dried powdered aerial parts of the plants were extracted sequentially with hexane, dichloromethane, methanol and water and tested for activity using a serial microdilution assay. Results were read every day over 120 h. All extracts except for the water extracts showed growth inhibitory activity against most isolates of the Fusarium spp. The most active were the methanol and hexane extracts of V. unguiculata and A. spinosus with minimum inhibitory concentration (MIC) values of <0·5 mg ml^?1 after 48 h against Fusarium spp. No inhibition of the Aspergillus spp. tested was observed, but conidium formation was stimulated on plates treated with plant extracts when visually compared to the growth controls. Conclusions: The results obtained from this study indicated that chemical constituents from these plant species may be developed as potential agrochemical fungicides. Significance and Impact of the Research: Food and feed are subject to infection by a variety of micro‐organisms that can induce spoilage and/or produce metabolites that are toxic to humans and animals. Extracts of V. unguiculata and A. spinosus were most active and maybe developed into environmentally friendly fungicides, which are affordable to rural farmers in developing countries.     

In vitro assays on the susceptibility of four species of nematophagous fungi to anthelmintics and chemical fungicides/antifungal drug

Using nematophagous fungi for the biological control of animal parasitic nematodes will become one of the most promising strategies in the search for alternative chemical drugs. The purpose of this study was to check the in vitro activity of four anthelmintics, four chemical fungicides and two antifungal drugs on the spore germination of nematophagous fungi: Duddingtonia flagrans (SF170), Arthrobotrys oligospora (447), Arthrobotrys superba (435) and Arthrobotrys sp. (PS011). A modified 24-well cell culture plate assay was conducted to evaluate the susceptibility of nematophagous fungi against drugs tested by calculating the effective middle concentrations (EC₅₀) of each tested drug to inhibit the germination of fungal spores. EC₅₀ ranged between 0·7 and 47·2 μg ml⁻¹ for fenbendazole, thiabendazole and ivermectin, except levamisole (546·5–4057·8 μg ml⁻¹). EC₅₀ of tested fungicides was 0·6–2·3 μg ml⁻¹ for carbendazim, 55·9–247·4 μg ml⁻¹ for metalaxyl, 24·4–45·2 μg ml⁻¹ for difenoconazole, and 555·9–1438·3 μg ml⁻¹ for pentachloronitrobenzene (PCNB). EC₅₀ of two antifungal drugs was 0·03–3·4 μg ml⁻¹ for amphotericin B and 0·3–10·9 μg ml⁻¹ for ketoconazole. The results showed that 10 tested drugs, except for levamisole and PCNB, had in vitro inhibitory effects on nematophagous fungi. The chlamydospores of D. flagrans had the highest sensitivity to nine tested drugs, except for ketoconazole.     

Biological activity of roots and aerial parts of Zinnia peruviana on pathogenic micro-organisms in planktonic state and biofilm forming

Microbial resistance to antibiotics affects the control of clinical infections and is a growing concern in global public health. One important mechanism whereby micro-organisms acquire resistance is biofilm formation. This context has led to the investigation of new antimicrobial substances from plants popularly used in folk medicine. In this work, we studied the antimicrobial and antibiofilm activity of Zinnia peruviana roots, ziniolide (major root metabolite) and aerial parts against Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa and Candida albicans. The minimum inhibitory and minimum microbicidal concentration and inhibition of biofilm production was determined. All Z. peruviana extracts showed antimicrobial activity, but that corresponding to the roots was the most active one. The best inhibitory and microbicidal activity was detected against Gram-positive bacteria (0·039–0·078 mg ml⁻¹). The acetonic extract from Z. peruviana leaves showed moderate activity against Gram-positive bacteria (0·625 mg ml⁻¹). Acetonic extract of Z. peruviana flowers showed weak activity (1·25–5 mg ml⁻¹). All the extracts tested showed inhibition of biofilm formation, as well as the ziniolide, however, roots and flowers extracts showed higher antibiofilm activity particularly against Staphylococcus, Listeria and Candida. The extracts tested may be a promising natural alternative for the control of microbial infections.     

GROWTH CHARACTERISTICS AND ANTIOXIDANT PROPERTIES OF THE BENTHIC DIATOM NAVICULA INCERTA (BACILLARIOPHYCEAE) FROM JEJU ISLAND,KOREA1

Benthic diatoms are a commonly used food source in shellfish aquaculture. Diatoms of the genus Navicula are the most abundant benthic diatoms occurring year‐round on the coast of Jeju Island, Korea. We isolated an axenic strain of N. incerta Grunow; estimated its growth characteristics under 27 different combinations of temperature, salinity, and nutrients; and determined its biochemical composition and antioxidant activities. The maximum specific growth rate (μ_max), defined as the increase in cell density per unit time, was 0.81–1.04 · d^?1, and the maximum cell density, 7.99 × 10⁵ cells · mL^?1, was reached at 0.88 · d^?1 μ_max, 20°C, 30 psu salinity, and F/2 nutrient concentration on day 12 of the culture period. The approximate cellular composition was as follows: 7.0 ± 0.04% protein, 1.7 ± 0.28% lipid, 12.8 ± 0.85% carbohydrate, 68.4 ± 0.09% ash, and 10.1 ± 0.44% moisture. The antioxidant properties of N. incerta were determined for various extracts. The rates of 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) free‐radical scavenging for Neutrase and methanol extracts were 81.6% and 62.8%, respectively. Flavourzyme extract had a superoxide‐scavenging rate of 57.7%. Kojizyme and Ultraflo extracts had nitric‐oxide‐scavenging rates of 42.2% and 40.6%, respectively, significantly higher than commercial antioxidants, such as α‐tocopherol and butylated hydroxytoluene (BHT). The metal‐chelating activities of the methanol, Neutrase, and Termamyl extracts were 68.5%, 45.2%, and 41.2%, respectively, four to six times higher than commercial antioxidants. The Termamyl extract showed the highest linoleic acid peroxidation inhibition, exceeding α‐tocopherol and on par with BHT.     

Detection of anti-tuberculosis activity in some folklore plants by radiometric BACTEC assay

Aims: The anti‐tubercular drugs are less effective because of the emergence of multi‐drug resistant (MDR) and extensively drug resistant (XDR) strains of M. tuberculosis, so plants being an alternative source of anti‐microbial compounds. The aim of this study was to investigate anti‐tuberculosis potential of the plants using Mycobacterium smegmatis as a rapid screening model for detection of anti‐mycobacterial activity and further to evaluate the active plants for anti‐tuberculosis activity against M. tuberculosis using radiometric BACTEC assay. Methods and Results: The 15 plants were screened for anti‐mycobacterial activity against M. smegmatis by the disk diffusion assay. The ethanolic extracts of Mallotus philippensis, Vitex negundo, Colebrookea oppositifolia, Rumex hastatus, Mimosa pudica, Kalanchoe integra and Flacourtia ramontchii were active against M. smegmatis in primary screening. The anti‐tuberculosis potential was identified in the leaves extracts of Mallotus philippensis by radiometric BACTEC assay. The ethanolic extract of M. philippensis showed anti‐tuberculosis activity against virulent and avirulent strains of M. tuberculosis H₃₇Rv and M. tuberculosis H₃₇Ra with minimum inhibitory concentration 0·25 and 0·125 mg ml^?1, respectively. The inhibition in growth index values of M. tuberculosis was observed in the presence of ethyl acetate fraction at a minimum concentration of 0·05 mg ml^?1. Conclusion: We found that BACTEC radiometric assay is a valuable method for detection of anti‐tuberculosis activity of the plant extracts. The results indicate that ethanolic extract and ethyl acetate fraction of M. philippensis exhibited significant anti‐mycobacterial activity against M. tuberculosis. Significance and Impact of the Study: These findings provide scientific evidence to support the traditional medicinal uses of M. philippensis and indicate a promising potential of this plant for the development of anti‐tuberculosis agent.     

In vitro anti‐enterovirus 71 activity of gallic acid from Woodfordia fruticosa flowers

Aims: The anti‐enterovirus 71 (EV71) activity of six Nepalese plants’ extracts and gallic acid (GA) isolated from Woodfordia fruticosa Kurz (family; Lythaceae) flowers were evaluated in Vero cells. Methods and Results: The anti‐EV71 activity of tested compounds was evaluated by a cytopathic effect reduction method. Our results demonstrated that flowers’ extracts of W. fruticosa exerted strong anti‐EV71 activity, with a 50% inhibitory concentration (IC₅₀) of 1·2 μg ml^?1 and no cytotoxicity at a concentration of 100 μg ml^?1, and the derived therapeutic index (TI) was more than 83·33. Rivabirin showed no antiviral activity against EV71. Furthermore, GA isolated from W. fruticosa flowers exhibited a higher anti‐EV71 activity than the extract of W. fruticosa flowers, with an IC₅₀ of 0·76 μg ml^?1 and no cytotoxicity at a concentration of 100 μg ml^?1, and the derived TI was 99·57. Conclusions: This study demonstrated that flower extracts of W. fruticosa possessed anti‐EV71 activity and GA isolated from these flowers showed stronger anti‐EV71 activity than that the extracts. Significance and Impact of the Study: Our results suggest that the GA from W. fruticosa flowers may be used as a potential antiviral agent.     

Exploring the Citrus Sour Rot pathogen: biochemical aspects,virulence factors,and strategies for disease management - a review

Citriculture is an important economic activity worldwide and for decades, this sector has been responsible for creating job opportunities. Currently, Brazil is the largest orange producer in the world, which contributes to the country's economy. However, citrus production has been facing several issues that compromise the quality of the fruits. For instance, several postharvest diseases occur during storage and transportation, directly harming product marketing. Green mold, blue mold, and sour rot are considered the most common postharvest citrus diseases. Citrus sour rot is less common; however, the disease can lead to a significant loss in high rainfall seasons. The fungus Geotrichum candidum is the causal agent of sour rot and its chemical and biochemical infection strategies are still little explored in citrus fruits. Several conventional control methods, including the application of fungicides, aim to contain the disease proliferation, but most of the commercially available fungicides are not efficient against sour rot. For this reason, other strategies have been studied for disease control, such as chemicals (e. g. essential oils or other natural products), biological agents used as biocontrol, and physical strategies. Despite its importance, few reviews have focused on sour rot disease. Here, we summarize the biochemical aspects of G. candidum, as well as the metabolites produced by this phytopathogen, the known virulence factors, and advances for disease management.     

Use of organic acids and salts to control postharvest diseases of lemon fruits in Egypt

Abstract

Postharvest diseases caused by Geotricum candidum (sour rot), Penicillium digitatum (green mould), and P. italicum (blue mould) are the most important negative factors affecting handling and marketing of citrus fruits in Egypt. The effect of organic acids (ascorbic, benzoic, citric and sorbic) as well as organic salts (potassium sorbate and sodium benzoate) were evaluated on the growth of causal agents and their disease incidence under in vitro and in vivo conditions. Complete inhibition was observed in the linear growth of all tested fungi when exposed to benzoic, citric and sorbic organic acids at concentrations of 4% and 2% of either sodium benzoate or potassium sorbate, respectively. Minimizing the tested concentration of organic acid down to 2%, the tested fungi fluctuated in their response such that only benzoic and sorbic acids could completely inhibit the growth of either P. digitatum or P. italicum only. Different organic acids and salts showed various levels of either protective or therapeutic effect for coated lemon fruits against mould infection whatever the time of their artificial inoculation under in vivo conditions. All treated fruits showed reduction in sour rot and green and blue mould diseases when compared with untreated fruits. Complete inhibition of mould incidence was obtained in coated lemon fruits with 4% of water or wax mixtures of sodium benzoate and potassium sorbate 24 hours before inoculation. Also, high reduction in mould incidence was observed in lemon fruits coated with the same concentration at 48 hours after inoculation under the same conditions. On the other hand, the tested organic acids showed a lesser effect on mould incidence. Moreover, they were more effective against mould incidence when dissolved in water than wax, that only 4% of water mixture of sorbic and benzoic acids showed 100% protection against mould incidence. Furthermore, the severity of infection records followed the same trend. The present findings demonstrate that potassium sorbate and sodium benzoate have potential as environmentally friendly products, nontoxic postharvest fungicides against sour rot, green and blue mould incidence of stored citrus fruits and could be suggested for commercial use in packing-houses in consideration to their wide consumption as safely food preservatives.     

Antimicrobial activity of crude extracts from mangrove fungal endophytes

The aim of this work was to select endophytic fungi from mangrove plants that produced antimicrobial substances. Minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) or minimal fungicidal concentrations (MFC) of crude extracts from 150 isolates were determined against potential human pathogens by a colorimetric microdilution method. Ninety-two isolates (61.3%) produced inhibitory compounds. Most of the extracts (28–32%) inhibited Staphylococcus aureus (MIC/MBC 4–200/64–200 μg ml⁻¹). Only two extracts inhibited Pseudomonas aeruginosa (MIC/MBC 200/>200 μg ml⁻¹). 25.5 and 11.7% inhibited Microsporum gypseum and Cryptococcus neoformans (MIC/MFC 4–200/8–200 μg ml⁻¹ and 8–200/8–200 μg ml⁻¹, respectively), while 7.5% were active against Candida albicans (MIC/MFC 32–200/32–200 μg ml⁻¹). None of the extracts inhibited Escherichia coli. The most active fungal extracts were from six genera, Acremonium, Diaporthe, Hypoxylon, Pestalotiopsis, Phomopsis, and Xylaria as identified using morphological and molecular methods. Phomopsis sp. MA194 (GU592007, GU592018) isolated from Rhizophora apiculata showed the broadest antimicrobial spectrum with low MIC values of 8–32 μg ml⁻¹against Gram-positive bacteria, yeasts and M. gypseum. It was concluded that endophytic fungi from mangrove plants are diverse, many produce compounds with antimicrobial activity and could be suitable sources of new antimicrobial natural products.     

Use of zero-valent iron biosand filters to reduce Escherichia coli O157:H12 in irrigation water applied to spinach plants in a field setting

Aims:  Zero‐valent iron (ZVI) filters may provide an efficient method to mitigate the contamination of produce crops through irrigation water. Methods:  A field‐scale system was utilized to evaluate the effectiveness of a biosand filter (S), a biosand filter with ZVI incorporated (ZVI) and a control (C, no treatment) in decontaminating irrigation water. An inoculum of c. 8·5 log CFU 100 ml^?1 of Escherichia coli O157:H12 was introduced to all three column treatments in 20‐l doses. Filtered waters were subsequently overhead irrigated to ‘Tyee’ spinach plants. Water, spinach plant and soil samples were obtained on days 0, 1, 4, 6, 8, 10, 13 and 15 and analysed for E. coli O157:H12 populations. Results:  ZVI filters inactivated c. 6 log CFU 100 ml^?1E. coli O157:H12 during filtration on day 0, significantly (P < 0·05) more than S filter (0·49 CFU 100 ml^?1) when compared to control on day 0 (8·3 log CFU 100 ml^?1). On day 0, spinach plants irrigated with ZVI‐filtered water had significantly lower E. coli O157 counts (0·13 log CFU g^?1) than spinach irrigated with either S‐filtered (4·37 log CFU g^?1) or control (5·23 log CFU g^?1) water. Soils irrigated with ZVI‐filtered water contained E. coli O157:H12 populations below the detection limit (2 log CFU g^?1), while those irrigated with S‐filtered water (3·56 log CFU g^?1) were significantly lower than those irrigated with control (4·64 log CFU g^?1). Conclusions:  ZVI biosand filters were more effective in reducing E. coli O157:H12 populations in irrigation water than sand filters. Significance and Impact of the Study:  Zero‐valent ion treatment may be a cost‐effective mitigation step to help small farmers reduce risk of foodborne E. coli infections associated with contamination of leafy greens.     

Molecular cloning and characterization of a novel laccase gene from a white-rot fungus Polyporus grammocephalus TR16 and expression in Pichia pastoris

Aims: To isolate a novel laccase gene from white‐rot fungus Polyporus grammocephalus TR16 and heterologous expression in Pichia pastoris. The characteristics of the heterologously expressed laccase are also studied. Methods and Results: Anchored PCR and 3′ RACE protocol were applied to obtain the full length of the laccase gene, which comprised 12 introns and an opening frame of 1769 bp. The deduced amino acid sequence of the laccase gene had an identity of 45–66% with the laccases reported previously. The cDNA was expressed in Pi. pastoris GS115 with native and α‐factor secretion signal peptides. The laccase activity obtained with the native signal peptide is threefold higher than that obtained with the α‐factor secretion signal peptide. The highest activity of the heterologously expressed laccase reached 893·3 U ml^?1, with its molecular mass estimated to be 65·4 kDa by SDS‐PAGE. The purified heterologously expressed laccase was stable at a pH range of 7·0–10·0. The optimum pH and temperature were 4·5 and 50°C, respectively; the K_m value for ABTS (3‐ethylbenzthiazoline‐6‐sulphonate) was 0·66 mmol l^?1. Conclusion: The novel laccase gene is cloned successfully and heterologously expressed in Pi. pastoris. Significance and Impact of the Study: A novel laccase gene isolated from a tropical fungus serves as a good source for pulp bleaching and wood processing.