Screening and identification of epiphytic yeasts with potential for biological control of green mold of citrus fruits

Epiphytic yeasts isolated from the surface of citrus fruits, harvested in several orchards in the Souss-Massa-Draa Valley, Agadir, Morocco, were in vivo screened for antagonistic activity against Penicillium digitatum, the causal agent of green mold of citrus. From a total of 245 yeast strains assessed for their biocontrol activity against P. digitatum, fifteen reduced the incidence of disease to less than 50%. The effectiveness of the best selected yeast strains showed that Pichia anomala (YT73), Debaryomyces hansenii (YT22) and Hanseniaspora guilliermondii (YT13) were the most effective, with a reduction of green mold incidence from 65 to ~80%, compared to the control. The identification of the fifteen selected yeast strains was carried out through an integrated approach including phenotypic and genotypic (sequencing of D1/D2 domain of 26S rDNA encoding gene) methods. These 15 selected were identified as: H. guilliermondii, D. hansenii, H. uvarum and P. anomala. The study of the dynamics of two of the best strains, H. guilliermondii and D. hansenii, showed that these strains can grow rapidly, by approximately 2 log units, in citrus fruit wounds. Such rapid growth in wounds indicates that these antagonist yeasts are excellent colonizers of citrus wounds and can thrive on citrus fruits as a substrate.     

Identification and characterization of chitin synthase genes in the postharvest citrus fruit pathogen Penicillium digitatum

In this study, we carried out the isolation and characterization of chitin synthase genes (CHS) of the main citrus fruit postharvest pathogen Penicillium digitatum. Using distinct sets of degenerate primers designed from conserved regions of CHS genes of yeast and filamentous fungi, PCR methods, and a DNA genomic library, five putative CHS genes (PdigCHSI, PdigCHSII, PdigCHSIII, PdigCHSV, and PdigCHSVII) were identified, isolated, sequenced, and characterized. Phylogenetic analyses, sequence identity, and domain conservation support the annotation as CHS. A very high sequence identity and strong synteny were found with corresponding regions from the genome of Penicillium chrysogenum. Gene expression of P. digitatum CHS genes during mycelium axenic growth, under oxidative and osmotic stress conditions, and during infection of citrus fruits was confirmed and quantified using quantitative RT-PCR (qRT-PCR). PdigCHSIII had the highest expression among the five genes by one order of magnitude, while PdigCHSII had the lowest. However, PdigCHSII was strongly induced coincident with conidial production, suggesting a role in conidiogenesis. The expression of PdigCHSI, PdigCHSIII, PdigCHSV, and PdigCHSVII was upregulated during infection of citrus fruit. PdigCHSV and PdigCHSVII coexpressed in most of the experiments carried out, and they are separated by a 1.77 kb intergenic region and arranged in opposite directions.     

The shelf life and effectiveness of granular formulations of Metschnikowia pulcherrima and Pichia guilliermondii yeast isolates that control postharvest decay of citrus fruit

Our overall objectives were to prepare commercially acceptable formulations of the postharvest biological control yeasts, Metschnikowia pulcherrima and Pichia guilliermondii, which have a long storage life and to determine the effectiveness of these formulations to control postharvest green and blue moulds on citrus fruit. Yeasts, grown on a cane molasses-based medium, were combined with talc or kaolin carriers and various adjuvants and the viability of yeast in 12 formulations was determined over a 6 month period. Formulation no. 11, containing talc, sodium alginate, sucrose, and yeast extract, for both yeasts had a significantly higher viable yeast cell content over a 6 month storage period. Among the formulations, three formulations (formulations no. 5, 6, and 11) were selected for additional in vivo testing because they had higher levels of viability amongst yeast cell populations during storage and were easier to resuspend remained in suspension more easily. These formulations were tested on Satsuma mandarin and grapefruit to control green and blue moulds. Formulations no. 5, 6, and 11 for both yeasts effectively controlled green mould, while only formulation no. 11 with either yeast isolate M. pulcherrima (isolate M1/1) or P. guilliermondii (isolate P1/3) effectively controlled both blue and green moulds.     

Biocontrol of a root rot of kale by <Emphasis Type="Italic">Muscodor albus</Emphasis> strain MFC2

Pythium ultimum is an oomycetous root rot pathogen that causes significant crop production losses on many crops including kale (Brassica oleracea), an economically important vegetable in Thailand. An endophytic fungus from Thailand designated Muscodor albus MFC2 controlled P. ultimum both in vitro and on kale seedlings grown under outdoor conditions via the production of volatile antibiotics. Ten-day old M. albus MFC2 PDA cultures killed P. ultimum in vitro. Thoroughly mixing three PDA plates of 10-day old M. albus MFC2 into a 500 g mixture of commercial soil and field soil did not adversely affect kale seed germination. The same amount of M. albus MFC2 could restore seedling emergence in P. ultimum inoculated soil to a level close to that of a non-infested control. In addition, M. albus MFC2 did not cause any disease symptoms, but rather seemed to promote the growth of kale in the presence or absence of P. ultimum for up to eight weeks after planting.     

Muscodor albus, a potential biocontrol agent against plant-parasitic nematodes of economically important vegetable crops in Washington State, USA

The fungus, Muscodor albus, was tested for nematicidal and nematostatic potential against four plant-parasitic nematode species with three different feeding modes on economically important vegetable crops in the Pacific Northwest. Meloidogyne chitwoodi, Meloidogyne hapla, Paratrichodorus allius, and Pratylenchus penetrans were exposed for 72 h to volatiles generated by M. albus cultured on rye grain in sealed chambers at 24 °C in the laboratory. In addition, the nematodes were inoculated into soil fumigated with M. albus, and incubated for 7 days prior to the introduction of host plants under greenhouse conditions. The mean percent mortality of nematodes exposed to M. albus in the chamber was 82.7% for P. allius, 82.1% for P. penetrans, and 95% for M. chitwoodi; mortality in the nontreated controls was 5.8%, 7%, and 3.9%, respectively. Only 21.6% of M. hapla juveniles died in comparison to 8.9% in controls. However, 69.5% of the treated juveniles displayed reduced motility and lower response to physical stimulus by probing, in comparison to the control juveniles. This is evidence of nematostasis due to M. albus exposure. The greenhouse study showed that M. albus caused significant reduction to all nematode species in host roots and in rhizosphere soil. The percent mortality caused by M. albus applied at 0.5% and 1.0% w/w in comparison to the controls was as follows: 91% and 100% for P. allius in the soil; 100% for P. penetrans in bean roots and soil; 85% and 95% for M. chitwoodi in potato roots, and 56% and 100% in the soil; 100% for M. hapla both in pepper roots and soil. In this study, M. albus has shown both nematostatic and nematicidal properties.     

Differentiation of pathogenic and nonpathogenic isolates of <Emphasis Type=Italic>Geotrichum candidum</Emphasis> sensu Suprapta et al. (1995) on citrus fruit based on PCR-RFLP analysis of rDNA ITS and PCR using specific primers designed in polygalacturonase genes

Pathogenic and nonpathogenic isolates of Geotrichum candidum sensu Suprapta et al. (1995) that affect citrus fruit are indistinguishable morphologically. In this work, differentiation of the two pathogenicity types based on the internal transcribed spacer regions of ribosomal DNA (rDNA-ITS) and polygalacturonase (PG) genes was attempted. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of rDNA ITS and PCR using specific primers to PG genes from each type could clearly differentiate the two pathogenicity types, and the profiles by PCR-RFLP of rDNA ITS genes corresponded with those by type-specific PCR of PG genes. These results indicate that the two pathogenic types can be differentiated at a molecular level and that PG genes are alternatively useful for distinguishing between the two types.     

Postharvest Imaging of Chlorophyll Fluorescence from Lemons Can Be Used to Predict Fruit Quality

We demonstrate the feasibility of assaying and predicting post-harvest damage in lemons by monitoring chlorophyll (Chl) fluorescence. Fruit quality was assayed using a commercial instrument that determines photosynthetic performance by imaging Chl fluorescence parameters under different irradiances. Images of Chl fluorescence from individual lemons reveal that photosynthesis is active throughout the post-harvest ripening process. Because photosynthesis is highly sensitive to biotic and abiotic stress, variations in Chl fluorescence parameters over the surface of a lemon fruit can be used to predict areas that will eventually exhibit visible damage. The technique is able to distinguish between mould-infected areas that eventually spread over the surface of the fruit, and damaged areas that do not increase in size during ripening. This study demonstrates the potential for using rapid imaging of Chl fluorescence in post-harvest fruit to develop an automated device that can identify and remove poor quality fruit long before visible damage appears.     

Muscodor albus MOW12 an Endophyte of Piper nigrum L. (Piperaceae) Collected from North East India Produces Volatile Antimicrobials

Muscodor albus MOW12, an endophytic fungus isolated from Piper nigrum in Mawlong, Meghalaya, India, resembles some cultural and hyphal characteristics of previous isolates of Muscodor sp. In addition, it possesses about 99 % similarity in its ITS rDNA with other M. albus isolates and thus is nicely centered within the genetic tree to other Muscodor spp. This xylariaceae fungus effectively inhibits and kills certain plant pathogenic fungi by virtue of a mixture of volatile compounds that it produces. The majority of these compounds were identified by gas chromatography/mass spectrometry as small molecular weight esters, alcohols, and acids. The main ester components of this isolate of M. albus in its volatile mixture are acetic acid, ethyl ester; propanoic acid, 2-methyl-, methyl ester and acetic acid, 2-methylpropyl ester. This appears to be the first report of any M. albus strain from India.     

The effect of temperature and duration of exposure of potato tuber moth (Lepidoptera: Gelechiidae) in infested tubers to the biofumigant fungus Muscodor albus

The endophytic fungus, Muscodor albus produces several volatile compounds (alcohols, esters, ketones, acids and lipids) that are biocidal for a range of organisms including plant pathogenic bacteria and fungi, nematodes and insects. We studied the effects of these volatiles on 3-day-old potato tuber moth larvae within infested tubers inside sealed chambers. The length of exposure to M. albus significantly affected mortality of larvae, calculated as percentage of larvae failing to survive to the adult stage. Exposure durations of 3, 7, or 14 days at 24 degrees C followed by incubation in fresh air at 27 degrees C until emergence resulted in mortalities of 84.2, 95.5 and 99.6%, respectively. However, the longer exposures also resulted in increased levels of carbon dioxide (CO(2)) that are unacceptable for tuber storage. Effects of M. albus on larval survival was also monitored at 10, 15 and 24 degrees C, using an exposure duration of 7 days followed by incubation in clean air at 27 degrees C until emergence. Mortality of larvae was sharply reduced at the lower temperatures resulting in 50.8, 76.8, and 95.4% mortality, respectively. Tuber storage conditions, especially cooling rates, are discussed with respect to using M. albus as a fumigant without simultaneously producing unacceptable (for tuber storage) levels of CO(2).     

The potential of the fungus, Muscodor albus, as a microbial control agent of potato tuber moth (Lepidoptera: Gelechiidae) in stored potatoes

Potato tuber moth (PTM), Phthorimaea operculella, is a serious pest of stored potato in most countries where potatoes are grown. Entomopathogens offer promise as alternatives to broad spectrum insecticides for management of this pest. The fungus Muscodor albus, which produces a mixture of antimicrobial volatile organic chemicals, was tested for its insecticidal activity against PTM. Adults and neonate larvae were exposed to volatiles generated by 15 or 30 g of M. albus rye grain culture plus water for 72 h in hermetically sealed 28.3 L chambers at 24 degrees C. Mean percent mortalities in adult moths exposed to 0, 15, and 30 g of fungal formulation were 0.9, 84.6, and 90.6%, respectively. Development to the pupal stage of PTM that were exposed as neonate larvae to 15 or 30 of M. albus culture was reduced by 61.8 and 72.8%, respectively, relative to controls.     

Isolation and metabolite production by Penicillium roqueforti,P. paneum and P. crustosum isolated in Canada

Penicillium roqueforti, P. crustosum and P. paneum grow on ensiled grain and recycled feed unless properly treated. The former two species occur also on cut lumber in Canada. These are known to produce a number of secondary metabolites including roquefortine. In cooler dairy production areas, including Scandinavia and North America, cattle toxicosis has been associated with silage contaminated by these fungi. We collected strains associated with cow or cattle toxicoses. The principal metabolites were determined making use of a new extraction method and analysis combining HPLC, LC/MS/MS, and LC/NMR. Penicillium roqueforti and P. crustosum required amino acid nitrogen for metabolite formation and their toxins were formed under conditions of low oxygen (20–30% saturation). Production of roquefortine C occurred on depletion of the available nitrogen and penitrem A on depletion of carbon source. Yield was reduced by excess carbon. Medium osmotic tension (a_w) affected metabolite production by the two species differently. Penicillium paneum was associated with ill-thrift of dairy cows and P. roqueforti was associated with more serious symptoms. Our data suggest a physiological basis for the common occurrence of roquefortine C in silage without serious consequences and the alternative, the presence of roquefortine C and toxicoses. The strain isolated from lumber was the best producer of the toxins studied. This is the first report of the toxigenic potential of P. roqueforti and P. paneum from Canada.     

Aroma production by spores ofPenicillium roqueforti on a synthetic medium

Summary The synthesis of 2-heptanone from the sodium salt of octanoic acid by spores of five strains ofPenicillium roqueforti was studied. The strains showed a high disparity in kinetic behavior. The one selected, which was originally isolated from blue cheese, had a good resistance to substrate inhibition along with a good apparent biotransformation yield (close to 60%). An activator was needed in the incubation medium. The loss of activity of aging spores was reduced by the activator compounds; ethanol exhibited the highest efficiency. When spores were produced on buckwheat seeds with a solid state fermentation technique, the medium itself was an activator source. When the biotransformation reaction was carried out in a stirred aerated fermentor, the volatile loss by air-stream stripping had to be taken into account. No ketone metabolism occurred with the strain used.     

Ectopic Expression of an <Emphasis Type=Italic>FT</Emphasis> Homolog from <Emphasis Type=Italic>Citrus</Emphasis> Confers an Early Flowering Phenotype on Trifoliate Orange (<Emphasis Type=Italic>Poncirus trifoliata</Emphasis> L. Raf.)

Citrus FT (CiFT) cDNA, which promoted the transition from the vegetative to the reproductive phase in Arabidopsis thaliana, when constitutively expressed was introduced into trifoliate orange (Poncirus trifoliata L. Raf.). The transgenic plants in which CiFT was expressed constitutively showed early flowering, fruiting, and characteristic morphological changes. They started to flower as early as 12 weeks after transfer to a greenhouse, whereas wild-type plants usually have a long juvenile period of several years. Most of the transgenic flowers developed on leafy inflorescences, apparently in place of thorns; however, wild-type adult trifoliate orange usually develops solitary flowers in the axils of leaves. All of the transgenic lines accumulated CiFT mRNA in their shoots, but there were variations in the accumulation level. The transgenic lines showed variation in phenotypes, such as time to first flowering and tree shape. In F₁ progeny obtained by crossing ‘Kiyomi’ tangor (C. unshiu × sinensis) with the pollen of one transgenic line, extremely early flowering immediately after germination was observed. The transgene segregated in F₁ progeny in a Mendelian fashion, with complete co-segregation of the transgene and the early flowering phenotype. These results showed that constitutive expression of CiFT can reduce the generation time in trifoliate orange.     

Isolation of mutants of the obligate methanotroph Methylomonas albus defective in growth on methane

A strain of Methylomonas albus BG8WM, a type 1 obligate methanotroph, which had been maintained for 2 ycars by serial passage on solid medium containing methanol as a carbon source was found to mutate at a frequency of 10^-5-10^-6 to resistance to dichloromethane (DCM^R), the parental strain BG8 did not give rise to DCM^R colonies. DCM^R strains were no longer capable of growth on methane as a carbon cource and exhibited greatly reduced or undetectable methane mono-oxygenase activity. The mutants fell into three groups on the basis of SDS-PAGE analysis of the polypeptide profiles of the particulate fraction of cell extracts. One or more of four polypeptides of Mr 70,000, 50,000, 25,000 and 23,000 were implicated as being components of the methane mono-oxygenase. Spontaneous reversion to growth on methane and sensitivity to dichloromethane occurred at a frequency of about 10^-8. The loss of methane mono-oxygenase activity was not associated with loss of the resident 55 kb plasmid.Abbreviations DCM^R dichloromethane-resistant - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - NMS nitrate minimal salts medium     

成年态南丰蜜橘试管嫁接育苗技术研究

为探索适合成年态南丰蜜橘[Citrus reticulata Blanco‘kinokuni’(Tanaka)H.H.Hu]的快速繁殖技术,对其试管茎尖微嫁接育苗进行研究。结果表明,最好的砧木是苦柚种子苗,以腹接方式的成活率最高。嫁接苗接种在MS+GA3 1 mg L–1+蔗糖75 g L–1的培养基中,暗培养7 d后转入光周期下培养,嫁接成活率达67.78%。不同移栽基质对嫁接苗的成活率影响不显著。嫁接苗与成年态南丰蜜橘再生芽在形态和POD、CAT及SOD同工酶分子表达上均无明显差异。这表明通过试管茎尖微嫁接技术可保持其遗传稳定性。     

Malate and malate-channel antibodies inhibit electrogenic and ATP-dependent citrate transport across the tonoplast of citrus juice cells

Citrus juice cells accumulate high levels of citric acid in their vacuoles when compared to other organic ions including malate. Uptake of citrate into tonoplast vesicles from Citrus juice cells was investigated in the presence of malate, and after incubation with antibodies raised against the vacuolar malate-specific channel of Kalancho? diagremontiana leaves. Antibodies against the vacuolar malate channel immunoreacted with a protein of similar size in tonoplast extracts from three Citrus varieties differing in citric acid content. Malate channel antibodies inhibited both delta MicroH(+)-dependent and delta MicroH(+)-independent ATP-dependent citrate transport, indicating common domains in both transport systems and to the malate-specific channel of Kalancho? diagremontiana leaves. Malate strongly inhibited electrogenic citrate transport, whereas ATP-dependent citrate uptake was less affected. Kinetic analysis of citrate transport in the presence of malate confirmed the existence of two citrate transport mechanisms and indicated that both citrate and malate share a common transport channel across the tonoplast of Citrus juice cells.     

The effects of Penicillium digitatum and Fusarium oxysporum rots on nutritional content of pawpaw (Carica papaya L.)

Investigations were carried out on the effects of Penicillium digitatum and Fusarium oxysporum on the nutritional value of pawpaw (Carica papaya). Decreases were observed in ash content, phosphorus, sodium, reducing sugars and ascorbic acid levels of fruits infected with P. digitatum, but increases in calcium and potassium content. In fruits infected by F. oxysporium, there were decreases in phosphorus, calcium, sodium ascorbic acid and reducing sugar levels; but the levels of ash content increased. The total protein level increased in the fruits infected with both fungi. These results revealed a reduction in fruit quality.