Inactivation of Penicillium italicum on kumquat via plasma-activated water and its effects on quality attributes.

Plasma-activated water (PAW) has good liquidity and uniformity and may be a promising candidate to inactivate Penicillium italicum and maintain the quality attributes of kumquat. In this study, the effect of plasma-activated water (PAW) on the viability of Penicillium italicum on kumquat and quality attributes of PAW-treated kumquats were then systematically investigated to elucidate the correlation between PAW and kumquat quality attributes. The effects of PAW on fruit decay, microbial loads, and firmness of postharvest kumquats during the 6-week storage were also investigated. The results showed that the viability of Penicillium italicum was notably inhibited by PAW on kumquats. Moreover, PAW did not significantly change the surface color of kumquats. No significant reductions in ascorbic acid, total flavonoid, and carotenoids were observed in kumquats after the PAW treatment. Results from nitrate and nitrite residue analyses showed that PAW did not leave serious nitrate and nitrite residues after treatment. The decay analysis results demonstrated that PAW has the potential to control kumquat decay and fungal contamination as well as maintain the firmness of postharvest kumquats throughout 6-week storage. Transmit electron microscope observation confirmed that PAW could cause the surface sculpturing in the skin cell wall of kumquat. The information obtained from this research may provide insight into the utilization of PAW to fight against fungal infection during the storage of citrus fruit.

Natural variations of TFIIAγ gene and LOB1 promoter contribute to citrus canker disease resistance in Atalantia buxifolia.

Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is one of the most devastating diseases in citrus industry worldwide. Most citrus cultivars such as sweet orange are susceptible to canker disease. Here, we utilized wild citrus to identify canker-resistant germplasms, and found that Atalantia buxifolia, a primitive (distant-wild) citrus, exhibited remarkable resistance to canker disease. Although the susceptibility gene LATERAL ORGAN BOUNDARIES 1 (LOB1) could also be induced in Atalantia after canker infection, the induction extent was far lower than that in sweet orange. In addition, three of amino acids encoded by transcription factor TFIIAγ in Atalantia (AbTFIIAγ) exhibited difference from those in sweet orange (CsTFIIAγ) which could stabilize the interaction between effector PthA4 and effector binding element (EBE) of LOB1 promoter. The mutation of AbTFIIAγ did not change its interaction with transcription factor binding motifs (TFBs). However, the AbTFIIAγ could hardly support the LOB1 expression induced by the PthA4. In addition, the activity of AbLOB1 promoter was significantly lower than that of CsLOB1 under the induction by PthA4. Our results demonstrate that natural variations of AbTFIIAγ and effector binding element (EBE) in the AbLOB1 promoter are crucial for the canker disease resistance of Atalantia. The natural mutations of AbTFIIAγ gene and AbLOB1 promoter in Atalantia provide candidate targets for improving the resistance to citrus canker disease.

Characterization of the bacterial communities of psyllids associated with Rutaceae in Bhutan by high throughput sequencing.

BACKGROUND: Several plant-pathogenic bacteria are transmitted by insect vector species that often also act as hosts. In this interface, these bacteria encounter plant endophytic, insect endosymbiotic and other microbes. Here, we used high throughput sequencing to examine the bacterial communities of five different psyllids associated with citrus and related plants of Rutaceae in Bhutan: Diaphorina citri, Diaphorina communis, Cornopsylla rotundiconis, Cacopsylla heterogena and an unidentified Cacopsylla sp. RESULTS: The microbiomes of the psyllids largely comprised their obligate P-endosymbiont 'Candidatus Carsonella ruddii', and one or two S-endosymbionts that are fixed and specific to each lineage. In addition, all contained Wolbachia strains; the Bhutanese accessions of D. citri were dominated by a Wolbachia strain first found in American isolates of D. citri, while D. communis accessions were dominated by the Wolbachia strain, wDi, first detected in D. citri from China. The S-endosymbionts from the five psyllids grouped with those from other psyllid taxa; all D. citri and D. communis individuals contained sequences matching 'Candidatus Profftella armatura' that has previously only been reported from other Diaphorina species, and the remaining psyllid species contained OTUs related to unclassified Enterobacteriaceae. The plant pathogenic 'Candidatus Liberibacter asiaticus' was found in D. citri but not in D. communis. Furthermore, an unidentified 'Candidatus Liberibacter sp.' occurred at low abundance in both Co. rotundiconis and the unidentified Cacopsylla sp. sampled from Zanthoxylum sp.; the status of this new liberibacter as a plant pathogen and its potential plant hosts are currently unknown. The bacterial communities of Co. rotundiconis also contained a range of OTUs with similarities to bacteria previously found in samples taken from various environmental sources. CONCLUSIONS: The bacterial microbiota detected in these Bhutanese psyllids support the trends that have been seen in previous studies: psyllids have microbiomes largely comprising their obligate P-endosymbiont and one or two S-endosymbionts. In addition, the association with plant pathogens has been demonstrated, with the detection of liberibacters in a known host, D. citri, and identification of a putative new species of liberibacter in Co. rotundiconis and Cacopsylla sp.

Antioxidant and biological activities of essential oil from Colombian Swinglea glutinosa(Blanco) Merr fruit

The objectives of this work were the study of the volatile chemical composition of essential oils (EO’s) from Swinglea glutinosa, as well as to evaluate their antioxidant, repellent and fumigant properties. The EO was obtained by hydrodistillation from the peel of the fruit, gathered in the city of Cartagena, Bolívar (Colombia). The volatile composition was analyzed by gas chromatography coupled to mass spectrometry (GC-MS). The major compounds found in S. glutinosaweregermacrene D (4.8%), limonene (5.2%),-terpineol (6.5%), -pinene (8.5%), nerolidyl acetate (9.8%), and trans-nerolidol (34.6%). S. glutinosashowed antioxidant potential (85.8%) (IC50=142.49 μg mL-1). The EO deployedrepellent activity against the Tribolium castaneumweevil at a concentration of 15.73 nL cm-1at 2 hours of exposure (72%), while the result for the commercial repellent was 50% at the same concentration. EO from S. glutinosadisplayed the best fumigant activity withLC50of 153.4 μg mL-1air. The essential oil from S. glutinosacan be considerated as a natural source of biocides and antioxidants.

Murraya paniculata and Swinglea glutinosa as Short-Term Transient Hosts of 'Candidatus Liberibacter asiaticus' and Implications for the Spread of Huanglongbing.

Murraya paniculata and Swinglea glutinosa are aurantioid hosts of the Asian citrus psyllid (ACP) Diaphorina citri, the principal vector of 'Candidatus Liberibacter asiaticus' (Las). Las is the pathogen associated with huanglongbing (HLB), the Asian form of which is the most devastating disease of Citrus species and cultivars (Rutaceae: Aurantioideae). M. paniculata is a common ornamental and S. glutinosa is grown as an ornamental, a citrus rootstock, and a hedgerow fence plant. Because of the uncertain status of these plants as reservoirs of Las, a series of cross-inoculation bioassays were carried out in different environments, using infected Valencia sweet orange (Citrus × aurantium) infected shoot tops as a source of inoculum and D. citri nymphs and adults reared on M. paniculata and S. glutinosa to inoculate pathogen-free Valencia orange plantlets. In contrast to sweet orange, Las was more unevenly distributed and reached much lower titers in M. paniculata and S. glutinosa. Infections in M. paniculata and S. glutinosa were also transient. Very few insects that successfully acquired Las from M. paniculata and S. glutinosa were able to transmit the pathogen to healthy citrus. Transmission rates were low from M. paniculata (1.0%) and S. glutinosa (2.0%) and occurred only in a controlled environment highly favorable to Las and ACP using 10-day-old adults that completed their life cycle on Las-positive plants. Our study showed that in HLB-endemic areas, M. paniculata and S. glutinosa can be deemed as epidemiologically dead-end hosts for Las and are not important alternative hosts of the pathogen for transmission to citrus. However, under a combination of conditions highly favorable to Las infection and transmission and in the absence of effective quarantine procedures, these plants could eventually serve as carriers of Las to regions currently free from HLB.

Isolation of limonoid compound (Hamisonine) from endophytic fungi Penicillium oxalicum LA-1 (KX622790) of Limonia acidissima L. for its larvicidal efficacy against LF vector, Culex quinquefasciatus (Diptera: Culicidae).

Upon screening for novel and potential biocompounds with larvicidal activities, we successfully isolated hamisonine (HMSN) a limonoid compound from endophytic fungi Penicillium oxalicum LA-1 of Limonia acidissima. The extracted compound structure was elucidated by spectral studies such as UV-vis spectroscopy, thin-layer chromatography, FTIR, LC-ESI-MS, 1H NMR, and 13C NMR upon comparing with the spectral data available in the literature. Further, the isolated HMSN was tested against III and IV instar Culex quinquefasciatus larvae. The outcome of this study clearly emphasize that the extracted compound HMSN possesses a stupendous larvicidal activity in a dose-dependent manner with the LC50 and LC90 values of 1.779 and 7.685 ppm against III instar larvae and 3.031 and 28.498 ppm against IV instar larvae of C. quinquefasciatus, respectively. Interestingly, the histological studies evidently showing the damage of peritrophic membrane and epithelial cells of testing mosquito larvae.

Chemical investigation of metabolites produced by an endophytic Aspergillus sp. isolated from Limonia acidissima.

Endophytic fungi are considered as a good source to produce important secondary metabolites with interesting bioactivities. In a continuation of our studies towards the search for environmentally friendly bioactive compounds from Sri Lankan flora, we investigated the secondary metabolites produced by the endophytic fungi Aspergillus sp. isolated from the seeds of the popular edible fruit Limonia acidissima L. of the family Rutaceae. The pure culture of the Aspergillus sp. was grown on potato dextrose broth media. After 4 weeks fermentation, fungal media were extracted with organic solvents. Chromatographic separation of the fungal extracts over silica gel, Sephadex LH-20 and RP-HPLC furnished flavasperone (1), rubrofusarin B (2), aurasperone A (3), fonsecinone D (4) and aurasperone B (5). Compounds 1-4 showed moderate activities in brine shrimp toxicity assay. This is the first report of the (13)C NMR data of compounds 4 and 5.

Three novel lineages of 'Candidatus Liberibacter africanus' associated with native rutaceous hosts of Trioza erytreae in South Africa.

Greening disease of citrus in South Africa is associated with 'Candidatus Liberibacter africanus' (Laf), a phloem-limited bacterium vectored by the sap-sucking insect Trioza erytreae (Triozidae). Despite the implementation of control strategies, this disease remains problematic, suggesting the existence of reservoir hosts to Laf. The current study aimed to identify such hosts. Samples from 234 trees of Clausena anisata, 289 trees of Vepris lanceolata and 231 trees of Zanthoxylum capense were collected throughout the natural distribution of these trees in South Africa. Total DNA was extracted from samples and tested for the presence of liberibacters by a generic Liberibacter TaqMan real-time PCR assay. Liberibacters present in positive samples were characterized by amplifying and sequencing rplJ, omp and 16S rRNA gene regions. The identity of tree host species from which liberibacter sequences were obtained was verified by sequencing host rbcL genes. Of the trees tested, 33 specimens of Clausena, 17 specimens of Vepris and 10 specimens of Zanthoxylum tested positive for liberibacter. None of the samples contained typical citrus-infecting Laf sequences. Phylogenetic analysis of 16S rRNA gene sequences indicated that the liberibacters obtained from Vepris and Clausena had 16S rRNA gene sequences identical to that of 'Candidatus Liberibacter africanus subsp. capensis' (LafC), whereas those from Zanthoxylum species grouped separately. Phylogenetic analysis of the rplJ and omp gene regions revealed unique clusters for liberibacters associated with each tree species. We propose the following names for these novel liberibacters: 'Candidatus Liberibacter africanus subsp. clausenae' (LafCl), 'Candidatus Liberibacter africanus subsp. vepridis' (LafV) and 'Candidatus Liberibacter africanus subsp. zanthoxyli' (LafZ). This study did not find any natural hosts of Laf associated with greening of citrus. While native citrus relatives were shown to be infected with Laf-related liberibacters, nucleotide sequence data suggest that these are not alternative sources of Laf to citrus orchards, per se.

[Population distribution and antimicrobial activities of endophytes in Toddalia asiatica].

OBJECTIVE: To study the population composition and antimicrobial activities of endophytes in medicinal plant Toddalia asiatica. METHODS: Endophytes were isolated from T. asiatica by using an exterior sterilization method, in combination with adding antimicrobial agents. Endophytes were classified and identified by morphological and molecular characters. Antimicrobial activities of endophytes were measured by using paper disc diffusion method. RESULTS: Three strains of endophytic bacteria, one strain of endophytic actinomyces and 82 strains of endophytic fungi were isolated from T. asiatica. Fusarium, Pestalotiopsis, and Aspergillus were the dominant populations in T. asiatica. Antimicrobial activities of these endophytes were measured against 30 pathogenic microbes, and 18 strains possess substantial inhibitory activities, of which 16 strains were endophytic fungi belonging to 11 genera. CONCLUSION: Endophytic strains with antimicrobial activities were obtained to explore the application of endophytic resources from T. asiatica.

New Meliolaceae from the Brazilian Atlantic forest 2: species on host families Annonaceae, Cecropiaceae, Meliaceae, Piperaceae, Rubiaceae, Rutaceae and Tiliaceae.

Continuing the study of black mildews in fragments of the Atlantic forest, three new species and five new records are described herein. Irenopsis luheae-grandiflorae, Meliola vicosensis and Meliola xylopia-sericiae are new species. Cecropia hololeuca, Piper gaudichaudianum and Trichilia lepidota are new hosts for Asteridiella leucosykeae, Asteridiella glabroides and Meliola trichiliae respectively. Asteridiella obesa and Meliola psychotriae var. chiococcae are reported for the first time from Brazil. The new species are described and illustrated based on light and scanning electron microscopy and tables with main characteristics of morphologically similar specimens with species collected in Viçosa are provided. Other species belonging to Meliolaceae collected on hosts belonging to the Annonaceae, Meliaceae and Tiliaceae in Brazil also were studied.

Different transcriptional response to Xanthomonas citri subsp. citri between kumquat and sweet orange with contrasting canker tolerance.

Citrus canker disease caused by Xanthomonas citri subsp. citri (Xcc) is one of the most devastating biotic stresses affecting the citrus industry. Meiwa kumquat (Fortunella crassifolia) is canker-resistant, while Newhall navel orange (Citrus sinensis Osbeck) is canker-sensitive. To understand the molecular mechanisms underlying the differences in responses to Xcc, transcriptomic profiles of these two genotypes following Xcc attack were compared by using the Affymetrix citrus genome GeneChip. A total of 794 and 1324 differentially expressed genes (DEGs) were identified as canker-responsive genes in Meiwa and Newhall, respectively. Of these, 230 genes were expressed in common between both genotypes, while 564 and 1094 genes were only significantly expressed in either Meiwa or Newhall. Gene ontology (GO) annotation and Singular Enrichment Analysis (SEA) of the DEGs showed that genes related to the cell wall and polysaccharide metabolism were induced for basic defense in both Meiwa and Newhall, such as chitinase, glucanase and thaumatin-like protein. Moreover, apart from inducing basic defense, Meiwa showed specially upregulated expression of several genes involved in the response to biotic stimulus, defense response, and cation binding as comparing with Newhall. And in Newhall, abundant photosynthesis-related genes were significantly down-regulated, which may be in order to ensure the basic defense. This study revealed different molecular responses to canker disease in Meiwa and Newhall, affording insight into the response to canker and providing valuable information for the identification of potential genes for engineering canker tolerance in the future.

Compost suppressiveness against Phytophthora spp. on Skimmia japonica and azalea.

Suppression of soil-borne plant diseases with composts has been widely studied. Composts have been found to be suppressive against several soil-borne pathogens in various cropping systems. Ornamental plants are generally cultivated in pots, allowing the use of suppressive substrates to control zoospore-producing pathogens, like Phytophthora sp. The objective of the present work was to assess compost suppressiveness against Phytophthora cinnamomi on Rhododendron spp., and against Phytophthora nicotianae, an emerging pathogen on Skimmia japonica. A municipal compost that showed a good suppressive activity in previous trials on vegetable crops was used. Compost was mixed at 10, 20 e 40% (v/v) with a commercial peat substrate, used as control. Substrates have been inoculated at 1g/l dosage of wheat and hemp kernels of Phytophthora spp. and after one week 15-20 plants were transplanted for each treatment in 2 liters volume pots and placed in greenhouse at 20 degrees C. A chemical control (Metalaxil-M) was also used. Diseased plants were assessed weekly after transplanting and above-ground biomass of plants was assessed at the end of the trials. Results showed a significant disease control when compost was used at 20-40% on S. japonica, without showing any phytotoxic effect. Disease suppression was shown at 40% on azalea, but compost was slightly phytotoxic on plants. The use of compost based substrates can be a suitable strategy for controlling soil-borne diseases on ornamentals, but results depend also on alkalinity tolerance of plants.

Fortunella margarita transcriptional reprogramming triggered by Xanthomonas citri subsp. citri.

BACKGROUND: Citrus canker disease caused by the bacterial pathogen Xanthomonas citri subsp. citri (Xcc) has become endemic in areas where high temperature, rain, humidity, and windy conditions provide a favourable environment for the dissemination of the bacterium. Xcc is pathogenic on many commercial citrus varieties but appears to elicit an incompatible reaction on the citrus relative Fortunella margarita Swing (kumquat), in the form of a very distinct delayed necrotic response. We have developed subtractive libraries enriched in sequences expressed in kumquat leaves during both early and late stages of the disease. The isolated differentially expressed transcripts were subsequently sequenced. Our results demonstrate how the use of microarray expression profiling can help assign roles to previously uncharacterized genes and elucidate plant pathogenesis-response related mechanisms. This can be considered to be a case study in a citrus relative where high throughput technologies were utilized to understand defence mechanisms in Fortunella and citrus at the molecular level. RESULTS: cDNAs from sequenced kumquat libraries (ESTs) made from subtracted RNA populations, healthy vs. infected, were used to make this microarray. Of 2054 selected genes on a customized array, 317 were differentially expressed (P < 0.05) in Xcc challenged kumquat plants compared to mock-inoculated ones. This study identified components of the incompatible interaction such as reactive oxygen species (ROS) and programmed cell death (PCD). Common defence mechanisms and a number of resistance genes were also identified. In addition, there were a considerable number of differentially regulated genes that had no homologues in the databases. This could be an indication of either a specialized set of genes employed by kumquat in response to canker disease or new defence mechanisms in citrus. CONCLUSION: Functional categorization of kumquat Xcc-responsive genes revealed an enhanced defence-related metabolism as well as a number of resistant response-specific genes in the kumquat transcriptome in response to Xcc inoculation. Gene expression profile(s) were analyzed to assemble a comprehensive and inclusive image of the molecular interaction in the kumquat/Xcc system. This was done in order to elucidate molecular mechanisms associated with the development of the hypersensitive response phenotype in kumquat leaves. These data will be used to perform comparisons among citrus species to evaluate means to enhance the host immune responses against bacterial diseases.

Micro-particle-induced X-ray emission mapping of elemental distribution in roots of a Mediterranean-type sclerophyll, Agathosma betulina (Berg.) Pillans, colonized by Cryptococcus laurentii.

The role of rhizosphere yeasts as plant nutrient-scavenging microsymbionts in resource-limited Mediterranean-type heathlands is unknown. This study, therefore, focused on quantitative elemental distribution within the roots of a medicinal sclerophyll, Agathosma betulina (Berg.) Pillans, grown under nutrient-poor conditions, and colonized by Cryptococcus laurentii. Micro-particle-induced X-ray emission (PIXE) was used to assess quantitative elemental distribution within the roots of A. betulina inoculated with viable C. laurentii, as well as within roots of control plants that received autoclaved yeast. To aid in the interpretation of heterogeneous elemental distribution patterns, apoplastic barriers (Casparian bands) in root tissues were located using fluorescence microscopy. In addition, root cross-sections were examined for endophytic C. laurentii using light and transmission electron microscopy (TEM). The average concentrations of P, Fe and Mn were significantly (P < 0.05) higher in roots of yeast-inoculated plants, compared to control plants. Casparian bands were observed in the exodermal cells of both treatments, and the presence of these bands was correlated with elemental enrichment in the epi/exodermal-outer cortical tissues. Light and TEM micrographs revealed that the yeast was not a root endophyte. This is the first report describing the role of a soil yeast as a plant nutrient-scavenging microsymbiont.

Evidence of symbiosis between the soil yeast Cryptococcus laurentii and a sclerophyllous medicinal shrub, Agathosma betulina (Berg.) Pillans.

The interaction between a common soil yeast, Cryptococcus laurentii, and a slow-growing medicinal plant adapted to low-nutrient soils, Agathosma betulina (Berg.) Pillans, was studied. C. laurentii CAB 578 was isolated from the rhizosphere of wild A. betulina, and liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis revealed that the yeast was capable of producing polyamines, such as cadaverine and spermine, while growing in vitro in a chemically defined medium. Since the exogenous application of polyamines are known to impact on root growth, these findings supported the results obtained when axenic cultures of A. betulina seedlings were inoculated with C. laurentii CAB 578 and cultivated for 5 months under glasshouse conditions. The presence of the yeast increased root growth by 51%. Using soil dilution plates, it was demonstrated that yeast numbers were greater in the vicinity of the roots than in the bulk soil. In addition, fluoromicroscopy, in combination with the fluorescent probes Fungolight and Calcofluor white, revealed the presence of metabolic active yeast colonies on the rhizoplane 5 months after initiation of the experimentation. The study provided evidence for a symbiosis between C. laurentii and A. betulina.

Rutaceae sampled from Germany, Malta, and Mallorca (Spain) are associated with AMF clustering with Glomus hoi Berch & Trappe.

Six Rutaceae species collected from natural habitats (Malta, Mallorca (Spain), and Tenerife (Spain)) and the Botanical Garden in Marburg were examined with respect to mycorrhizal structures and fungal identity. All species have the same gross colonization pattern of arbuscular mycorrhiza (AM) with distinct intracellular and intercellular phases but show remarkable differences in details, especially in terms of the extent of the intracellular phase. The associated AM fungi, identified using molecular methods, cluster together with Glomus hoi Berch & Trappe, although the plants were collected from very distant locations.