Analysis of endophytic and rhizosphere bacterial diversity and function in the endangered plant Paeonia ludlowii.

Paeonia ludlowii is indigenous to Tibet and has an important ecological and economic value in China. In Tibet, P. ludlowii has been used in folk medicine with relative success. Plant microbial endophytes play an important role in plant growth, health and ecological function. The diversity of endophytic bacteria associated with P. ludlowii remains poorly understood. In this study, the structure of the endophytic bacterial communities associated with different tissues, including fruits, flowers, leaves, stems, and roots, and rhizosphere soils was analyzed with Illumina MiSeq sequencing of bacterial 16S rDNA. A total of 426,240 sequences and 4847 operational taxonomic units (OTUs) were obtained. The OTUs abundance of roots was higher than that of other tissues; however, the OTUs abundance was similar among different deep soil samples. In the plant tissues, Cyanobacteria was the most abundant bacterial phylum, followed by Proteobacteria; however, the most abundant phyla were Proteobacteria and Acidobacteria in soil samples from three different layers. In addition, the diversity and richness of the microorganisms in the soil were very similar to those in roots but higher than those in other tissues of P. ludlowii. Predictive metagenome analysis revealed that endophytic bacteria play critical functional roles in P. ludlowii. This conclusion could facilitate the study of the ecological functions of endophytic bacteria and their interactions with P. ludlowii to analyze the reasons why this important medicinal plant is becoming endangered.

Complete genome sequence of an alternavirus from the phytopathogenic fungus Fusarium incarnatum.

A novel double-stranded RNA (dsRNA) mycovirus, named "Fusarium incarnatum alternavirus 1" (FiAV1), was found in Fusarium incarnatum strain LY003-07, the causal agent of peony root rot. The complete genome of FiAV1 is composed of three dsRNA segments, dsRNA1 (3548 nt), dsRNA2 (2514 nt), and dsRNA3 (2498 nt), with one open reading frame on each of their positive-sense strands. As found in other viruses of the proposed family Alternaviridae, the positive-sense strand of each genomic dsRNA of FiAV1 has a poly(A) tail and the 5'-terminal nonamer sequence 5'-GGCTGTGTG-3'. Based on a multiple sequence alignment and phylogenetic analysis based on the RdRps amino acid sequence, FiAV1 is suggested to be a new strain of a potential new species, for which the name "Fusarium alternavirus 1" is proposed, that includes Fusarium poae alternavirus 1 (FpAV1) and Fusarium graminearum alternavirus 1 (FgAV1/AH11) of the proposed family "Alternaviridae". This is the first report of a mycovirus of the proposed family "Alternaviridae" that infects F. incarnatum.

PlWRKY13: A Transcription Factor Involved in Abiotic and Biotic Stress Responses in Paeonia lactiflora.

Many members of the WRKY family regulate plant growth and development. Recent studies have shown that members of the WRKY family, specifically WRKY13, play various roles in the regulation of plant stress resistance. To study the function of WRKY family members in peony, the PlWRKY13 gene (KY271095) was cloned from peony leaves. Sequence analysis and subcellular localization results revealed that PlWRKY13 has no introns, belongs to the type IIc subgroup of the WRKY family, and functions in the nucleus. The expression pattern of PlWRKY13 was analysed via real-time quantitative RT-PCR (qRT-PCR), which showed that the expression of PlWRKY13 was induced by four types of abiotic stress, low-temperature, high-temperature, waterlogging and salt stress, and was positively upregulated in response to these stresses. In addition, the expression of PlWRKY13 tended to first decrease and then increase after infection with Alternaria tenuissima. Virus-induced gene silencing (VIGS) technology was used to explore the function of PlWRKY13 in the resistance of Paeonia lactiflora to fungal infection further, and the results showed that PlWRKY13-silenced plants displayed increased sensitivity to A. tenuissima. The infection was more severe and the disease index (DI) significantly greater in the PlWRKY13-silenced plants than in the control plants, and the expression of pathogenesis-related (PR) genes was also significantly altered in the PlWRKY13-silenced plants compared with the control plants. The contents of the endogenous hormones jasmonic acid (JA) and salicylic acid (SA) were measured, and the results showed that the JA content increased gradually after infection with A. tenuissima and that JA may play an active role in the resistance of P. lactiflora to pathogen infection, while the SA content decreased after PlWRKY13 silencing. The contents of the two hormones decreased overall, suggesting that they are related to the transcription of PlWRKY13 and that PlWRKY13 may be involved in the disease-resistance pathway mediated by JA and SA. In summary, the results of our study showed that PlWRKY13 expression was induced by stress and had a positive effect on the resistance of P. lactiflora to fungal infection.

Paenibacillus paeoniae sp. nov., a novel endophytic bacterium isolated from leaf of Paeonia lactiflora Pall.

A Gram-stain-positive, aerobic, rod-shaped, endospore-forming bacterium, designated strain M4BSY-1T, was originally isolated from a surface-sterilized leaf of Paeonia lactiflora Pall. in Guizhou, China. The bacterium was characterized by a polyphasic approach to determine its taxonomic position. 16S rRNA gene sequence comparison revealed that strain M4BSY-1T belongs to the genus Paenibacillus and most closely to Paenibacilluspinioli NB5T (98.31 % similarity). Neither substrate nor aerial mycelia formed, and no diffusible pigments were observed on the media tested. Strain M4BSY-1T grew in the pH range 7.0-13.0 (optimum, 7.0-8.0), at temperatures between 10-37 °C (optimum, 30 °C) and at 0-5 % (w/v) NaCl (optimum, 1-2 %). The predominant menaquinone was MK-7. The major fatty acids were anteiso-C15 : 0, C16 : 0 and iso-C16 : 0. The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unidentified unknown aminophospholipids and three unidentified phospholipids. The DNA G+C content was 48.8 mol%. According to the phylogenetic, phenotypic and chemotaxonomic evidence, strain M4BSY-1T was clearly distinguishable from other species with validly published names in the genus Paenibacillus and should therefore be classified as a novel species, and we suggest the name Paenibacillus paeoniae sp. nov. The type strain is M4BSY-1T (=KCTC 33997T=CGMCC 1.13667T).

Culturable Endophytes Diversity Isolated from Paeonia ostii and the Genetic Basis for Their Bioactivity.

Paeonia ostii is known for its excellent medicinal values as Chinese traditional plant. To date, the diversity of culturable endophytes associated with P. ostii is in its initial phase of exploration. In this study, 56 endophytic bacteria and 51 endophytic fungi were isolated from P. ostii roots in China. Subsequent characterization of 56 bacterial strains by 16S rDNA gene sequence analysis revealed that nine families and 13 different genera were represented. All the fungal strains were classed into six families and 12 genera based on ITS gene sequence. The biosynthetic potential of all the endophytes was further investigated by the detection of putative polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes. The PCR screens were successful in targeting thirteen bacterial PKS, five bacterial NRPS, ten fungal PKS and nine fungal NRPS gene fragments. Bioinformatic analysis of these detected endophyte gene fragments facilitated inference of the potential bioactivity of endophyte bioactive products, suggesting that the isolated endophytes are capable of producing a plethora of secondary metabolites. These results suggest that endophytes isolated from P. ostii had abundant population diversity and biosynthetic potential, which further proved that endophytes are valuable reservoirs of novel bioactive compounds.Paeonia ostii is known for its excellent medicinal values as Chinese traditional plant. To date, the diversity of culturable endophytes associated with P. ostii is in its initial phase of exploration. In this study, 56 endophytic bacteria and 51 endophytic fungi were isolated from P. ostii roots in China. Subsequent characterization of 56 bacterial strains by 16S rDNA gene sequence analysis revealed that nine families and 13 different genera were represented. All the fungal strains were classed into six families and 12 genera based on ITS gene sequence. The biosynthetic potential of all the endophytes was further investigated by the detection of putative polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes. The PCR screens were successful in targeting thirteen bacterial PKS, five bacterial NRPS, ten fungal PKS and nine fungal NRPS gene fragments. Bioinformatic analysis of these detected endophyte gene fragments facilitated inference of the potential bioactivity of endophyte bioactive products, suggesting that the isolated endophytes are capable of producing a plethora of secondary metabolites. These results suggest that endophytes isolated from P. ostii had abundant population diversity and biosynthetic potential, which further proved that endophytes are valuable reservoirs of novel bioactive compounds.

Botrytis euroamericana, a new species from peony and grape in North America and Europe.

A novel species of Botrytis isolated from peony in Alaska, USA, and grape in Trento District, Italy, was identified based on morphology, pathogenicity, and sequence data. The grape and peony isolates share sequence homology in the glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat shock protein 60 (HSP60), DNA-dependent RNA polymerase subunit II (RPB2), and necrosis- and ethylene-inducing protein 1 and 2 (NEP1 and NEP2) genes that place them in a distinct group closely related to B. aclada, a globally distributed pathogen of onions. Genetic results were corroborated with morphological and pathogenicity trials that included two isolates of B. cinerea and two isolates of B. paeoniae from peony in Alaska and one isolate of B. aclada. The authors observed differences in colony and conidia morphology and ability to cause lesions on different host tissues that suggest that the grape and peony isolates represent a distinct species. Most notably, the grape and peony isolates did not colonize onion bulbs, whereas B. aclada readily produced lesions and prolific sporulation on onion tissue. The new species Botrytis euroamericana is described herein.

[Rhizosphere microorganism evidence on geo-authentic features of traditional Chinese medicine from Moutan(Paeonia suffruticosa)].

Paeonia suffruticosa also named Moutan that cultivated in five geographic regions during different growth stages were chosen in this study. Biolog and 454 pyrosequencing technology were used to detect the whole microbial activity and fungal diversity for exploring the relationship between the geo-authentic features of the medicinal plant and the rhizosphere microorganism. The results suggest that the value of average well color development(AWCD) from the rhizosphere soil of P. suffruticosa in the five regions at the four growth stage have an increasing tendency. 9 703 operational taxonomic unit(OTU) were obtained from 272 463 high quality sequences according to the similarity of 97% by the pyrosequencing. Fungi in five phyla, twenty-two classes, seventy orders, one hundred and thirty-nine families and two hundred and sixty-six genera were detected in the five regions excluding twelve percent to fifty-eight percent unidentified fungi. They were divided into four branches, i.e. Blastocladiales, Chytridiomycota, Dikarya and Glomeromycetes. Twenty-four genera such as Leptosphaeria were found in the five regions while six genera such as Curvularia were only detected in the geo-authentic regions. The dominant genera were Guehomyces, Exophiala and Fusarium in geo-authentic regions, whereas genus Leptosphaeria, Cryptococcus, Exophiala, Fusarium and Ceratobasidium in non-authentic regions. The results from principal co-ordinates analysis (PCoA) showed that the fungi formations were similar in Tongling and Nanling region at four different growth stages, and the same in Heze at the leaf bud and fruiting stage. According to heatmap analysis, Tongling and Nanling region showed a close similarity in fungal community structures on phylogenetic analysis, while Bozhou, Heze and Luoyang showed the same. In brief, the whole microbial activity was higher in geo-authentic regions than the non-authentic. Fungi in rhizosphere soil of the medicinal peony presented diversity and region specificity. We found not only the abundant new species in the five regions, but also the phylogenetic similarity in the geo-authentic regions.

[Isolation, identification and inhibition activity of endophytes in Fengdan].

Endophytes were isolated and purified from the roots of medicinal plant Fengdan also known as Paeonia suffruticosa from Tongling region, Anhui province, China. Morphology and molecular biology methods were applied to indentify the endophyte strains. And methods of growth rate and filtering paper were also used for studying antibacterial/antifungal effects of the strains. As a result, 129 endophyte strains were isolated. Fifty-eight endophytic fungi strains were identified as 6 species in 4 genera and the dominant genus was Fusarium. Seventy-one endophytic bacteria strains were identified as 9 species in 3 genera and the dominant genus was Bacillus. Inhibitory diameter with endophytic fermenting liquid of Pseudomonas chlororaphis, F. nematophilum and B. megaterium from P. suffruticosa against Staphyloccocus aureus, Escherichia coli and B. subtilis reached 25.0,20.2,24.0 mm respectively. The inhibition rate of endophytic fermenting liquid from F. nematophilum against Penicillium sp. and Colletotrichum dematium reached 90.6% and 83.3%, respectively. The inhibition effect of P. chlororaphis against F. oxysporum f. sp. niveum and Mucor sp. was good and the antifungal rate reached 80.0% and 84.9%, respectively. P. suffruticosa in Tongling region contains abundant endophytes. P. chlororaphis and F. nematophilum are valuable species as starting strain about microbicide.

[Effects of growth years of Paeonia lactiflora on bacterial community in rhizosphere soil and paeoniflorin content].

To explore the relationship between microecological environment and Paeonia lactiflora the effects of growth years of P. lactillora on rhizosphere bacterial communities were studied by PCR-DGGE and the paeoniflorin content determined by HPLC. Results showed that the soil pH increased with growing years of P. lactillora. In the fourth year, soil pH and enzyme activity reached the highest level, while organic matter content was the lowest. The bacterial diversity had a positive correlation with growing years varied from 3.38 to 3.61. Sequencing results demonstrated that Gammaproteobacteria, llphaproteobacteria, Actinobacteria, Acidobacte- ria and Firmicutes were predominant bacteria kinds in the soil of P. lactillora. Gammaproteobacteria was only detected in the bulk soil, while llphaproteobacteria, Acidobacteria G1l, Actinobacteria were only in the rhizosphere soil and the bacterial community among different growing years were similar except few species. HLPC results showed that paeoniflorin content was 3.26%, 3.30%, 3.36%, 3.41% separately from one to four-year-old P. lactiflora with an upward trend. The correlation analysis indicated that the paeoniflorin content had a positive correlation with soil pH and bacterial diversity, conversely, had a negative correlation with organic matter con- tent. During the growth years the rhizosphere bacterial diversity increased without changes of predominant bacteria and the paeoniflorin content increased without significant differences while its production increased significantly, which was different from the plants showing replanting diseases. This is in line with the farming practice choosing 4-year-old P. lactllora, but not the 1-3 year old one. In addition, the accumulation of paeoniflorin is closely related to soil pH, organic matter content and bacteria diversity, confirming that the geoherblism of P. lactiflora is closely related with microbial environment in the soil.

[Identification and secondary metabolites of endophytic fungal strain PR35 from Paeonia delavayi].

OBJECTIVE: To identify the endophytic fungal strain PR35 separated from Paeonia delavayi and study chemical constituents of its secondary metabolites. METHOD: The fungal strain PR35 was identified by morphological observation and ITS rDNA sequence analysis. Various chromatographic methods were adopted to separate and purify its secondary metabolites, and their structures were identified by physiochemical properties and spectral data RESULT: The fungal strain PR35 was identified as Trichoderma longibrachiatum. Five compounds were separated from fermentation products of fungal strain PR35 and identified as 1-(2,6-dihydroxyphenyl)-3-hydroxybutan-1-one (1), 1-(2,6-dihydroxypheny) propan-1-one (2), 1-(2,4,6-trihydroxyphenyl) butan-1-one (3), 4-methoxy-1-naphthol (4), and cerevisterol (5). Among them, compounds 1-3 showed notable antifungal activities against Botrytis cinerea, Fusarium avenaceum and Hormodendrum compactum. CONCLUSION: The endophytic fungus T. longibrachiatum was separated from the plant P. delavayi for the first time. Five compounds were first separated from endophytic fungus of P. delavayi. Among them, compound 4 was separated from microbial fermentation products for the first time.

Sesquiterpenoids from the endophytic fungus Trichoderma sp. PR-35 of Paeonia delavayi.

A new bisabolane-type sesquiterpene, trichoderic acid (1), and a new acorane-type sesquiterpene, 2ß-hydroxytrichoacorenol (2), along with three known compounds, cyclonerodiol (3), cyclonerodiol oxide (4), and sorbicillin (5), were isolated from the culture broth of Trichoderma sp. PR-35, an endophytic fungus isolated from Paeonia delavayi. Their structures were elucidated on the basis of their IR, MS, and 1D- and 2D-NMR analyses. The antibacterial and antifungal activities of 1-5 towards various types of bacteria and fungi were tested. Most of the compounds showed moderate or weak antimicrobial activities in an agar-diffusion assay.

Evaluating the Comprehensive Performance of Herbaceous Peonies at low latitudes by the Integration of Long-running Quantitative Observation and Multi-Criteria Decision Making Approach.

Enlarging the planting area of economic plants, such as the "Southward Planting of Herbaceous Peony" (Paeonia lactiflora. Pall), is significant for improving people's lives. Peony is globally known as an ornamental because of gorgeous flowers and is mainly cultivated in the temperate regions with relatively cool and dry climates in the Northern Hemisphere. Promoting the landscape application of peony to the lower latitude regions is difficult because of the hot-humid climate. In this study, 29 northern peony cultivars and a unique Chinese southern peony, 'Hang Baishao', were introduced to Hangzhou, located in the central subtropics. Annual growth cycles, resistances and dormancy durations were measured, and crossbreeding between the southern and northern peonies was performed for six years, from 2012 to 2017. Based on data collected from the long-running quantitative observation (LQO), a multi-criteria decision making (MCDM) system was established to evaluate the comprehensive planting performance of these 30 cultivars in the central subtropics. 'Qihua Lushuang', 'Hang Baishao' and 'Meiju' were highly recommended, while 'Zhuguang' and 'Qiaoling' were scarcely recommended for the Hangzhou landscape. This study highlights the dependability and comprehensiveness of integrating the LQO and MCDM approaches for evaluating the introduction performance of ornamental plants.

Aspergilolide, a steroid lactone produced by an endophytic fungus Aspergillus sp. MBL1612 isolated from Paeonia ostii.

A new steroid lactone aspergilolide (1), and nine known compounds helvolic acid (2), verruculogen (3), tryprostatin B (4), 13-oxofumitremorgin B (5), fumitremorgin C (6), demethoxy fumitremorgin C (7), terezine D (8), aszonalenin (9), 12, 13-dihydroxy-fumitremorgin C (10) from cultures of the endophytic fungus Aspergillus sp. MBL1612. Their chemical structures were determined by a series of extensive spectroscopic methods. All of the compounds were isolated from this genus for the first time. The cytotoxicity against five human cancer cell lines of new compound were detected.

Aspergilates A to E, second metabolites from Aspergillus sp. isolated from Paeonia ostii.

Five novel globoscinic acid derivatives, aspergilates A-E (1-5) have been isolated from endophytic fungus Aspergillus sp. derived from Paeonia ostii. The structures of aspergilates A-E were determined by in-depth analyses of NMR spectra, HRESIMS and CD experiments. The absolute configurations of compounds 2-5 were determined by the ECD calculations based on quantum chemistry. Cytotoxic activities against five selected tested tumor cell lines of 1-5 were evaluated.

Diversity of endophytic fungi of Paeonia lactiflora Pallas and screening for fungal paeoniflorin producers.

Endophytic fungi from Paeonia lactiflora Pallas, which is mainly distributed in China, were characterized and screened to identify those capable of producing paeoniflorin. A total of 101 isolates obtained from the roots, stems and leaves of P. lactiflora were grouped into 16 fungal taxa based on morphological traits and internal transcribed spacers sequences, indicating that endophytic fungi of P. lactiflora are abundant and diverse. The dominant endophytic fungi were Aspergillus, Alternaria and Penicillium. More fungi were recovered from leaves than from roots and stems. The similarity index was highest between the stems and leaves (0.733), followed by the roots and leaves (0.615) and the stems and roots (0.563). Analyses of the fermentation extracts of 22 endophytic fungi by high-performance liquid chromatography and mass spectrometry revealed that three strains (R12, Alternaria tenuissima; S4, Aspergillus flavus; and R17 Penicillium commune) were able to produce paeoniflorin. Among the paeoniflorin-producing fungi, the yield of paeoniflorin from A. flavus S4 was 342.4 µg/L, and this strain could be used as a candidate for the industrial production of paeoniflorin.

Polyoxygenated Cyclohexenoids with Promising α-Glycosidase Inhibitory Activity Produced by Phomopsis sp. YE3250, an Endophytic Fungus Derived from Paeonia delavayi.

Seven new polyoxygenated cyclohexenoids, namely, phomopoxides A-G (1-7), were isolated from the fermentation broth extract of an endophytic fungal strain Phomopsis sp. YE3250 from the medicinal plant Paeonia delavayi Franch. The structures of these compounds were established by spectroscopic interpretation. The absolute configurations of compounds 1 and 4 were confirmed by X-ray crystallographic analysis and chemical derivative approach. All isolated compounds showed weak cytotoxic activities toward three human tumor cell lines (Hela, MCF-7, and NCI-H460) and weak antifungal activities against five pathogenic fungi (Candida albicans, Aspergillus niger, Pyricularia oryzae, Fusarium avenaceum, and Hormodendrum compactum). In addition, compounds 1-7 showed a promising α-glycosidase inhibitory activity with IC50 values of 1.47, 1.55, 1.83, 2.76, 2.88, 3.16, and 2.94 mM, respectively, as compared with a positive control of acarbose (IC50 = 1.22 mM).

Diversity and communities of culturable endophytic fungi from different tree peonies (geoherbs and non-geoherbs), and their biosynthetic potential analysis.

To know more about the potential roles of endophytic fungi in the formation mechanism of Daodi medicinal material, diversity and communities of culturable endophytic fungi in three types of tree peonies were investigated. Endophytic fungi of three types of tree peonies were isolated and identified. The diversity was analyzed. Bayesian trees constructed by MrBayes 3.2.6 after phylogenetic analysis of the ITS sequences. The endophytic fungi potential for synthesis of natural products was assessed by means of detecting NRPS and PKS gene sequences. In total, 364 endophytic fungi isolates representing 26 genera were recovered from Paeonia ostii 'Feng Dan', Paeonia ostii 'Luoyang Feng Dan', and Paeonia suffruticosa 'Luoyang Hong'. More culturable endophytic fungi appeared in P. suffruticosa 'Luoyang Hong' (206) compared with P. ostii 'Feng Dan' (60) and P. ostii 'Luoyang Feng Dan' (98). The fungal community of P. ostii 'Feng Dan' had the highest richness and diversity. PKSs and NRPS detection rates of endophytic fungi from P. ostii 'Feng Dan' are both the highest among the three types of tree peonies. Results indicate that endophytic fungus is an important factor of Daodi Cortex Moutan forming, and endophytic fungi in peony are related to genuineness of Cortex Moutan.

Illumina-based analysis of endophytic bacterial diversity of tree peony (Paeonia Sect. Moutan) roots and leaves.

Diverse communities of bacteria inhabit plant tissues and those bacteria play a crucial role for plant health and growth. Tree peony (Paeonia Sect. Moutan) is known for its excellent ornamental and medicinal values as Chinese traditional plant, but little is known about its associated bacterial community under natural conditions. To examine how endophytic bacteria in tree peony vary across tissues and cultivars, PCR-based Illumina was applied to reveal the diversity of endophytic bacteria in tree peony. A total of 149,842 sequences and 21,463 operational taxonomic units (OTUs) were obtained. The OTU abundance of roots was higher than leaves across other three cultivars except for 'Kinkaku' and 'Luoyanghong'. The community was composed of five dominant groups (Proteobacteria, Firmicutes, Bacteroidetes, Acidobacteria and Actinobacteria) in all samples. Endophytic bacteria community structures had changed in leaves and roots. Sequences of Pseudomonas and Enterobacteriaceae were prevalent in root samples, whereas Succinivibrio and Acinetobacter were the dominant genus in leaf samples. Otherwise, the distribution of each dominant genus among the 5 cultivars was either varied. These findings suggested that both plant genotype and tissues contribute to the shaping of the bacterial communities associated with tree peony.

[Isolation, classification and antimicrobial activity of endophytic actinomycetes from plant leaves].

Leave samples of Paeonia lactiflora and Trifalium repens were collected from Sichuan Province and Beijing respectively to study their endophytic actinmycetes. After a well-established surface-sterilized procedure, the samples were plated on agar media of TWYE, HV, YECD, NA and WA, followed by incubation at 28 degrees C for 2 - 4 weeks. With the help of light microscope, 15 actinomycetes strains were isolated from the plates. Comparison of cultural features and fingerprinting analyses of BOX-PCR products were performed to cluster the isolates, with the result that all the strains were assigned to 12 different genotypes, half of them from Paeonia lactiflora and half from Trifalium repens. A combination of morphological and 16S rRNA gene sequence data showed that except strains C4 and C5, which belonged to Pseudonocardia, 13 of the isolates were streptomycetes. Most isolates share high 16S rRNA gene sequence similarities to known type strains. However, strain C12 shares low values, 96.6% in maximum, and strain C5 shares 97.9% in maximum; more assays are needed to ascertain their taxonomic positions. In the tests of antimicrobial activity against 7 bacteria, 3 fungi and 1 yeast, 11 isolates were positive in one or more tests, and 55% of the positive ones could inhibit the growth of Rhizoctonia solani, a significant pathogen of plants.

Molecular identification of Botrytis cinerea, Botrytis paeoniae and Botrytis pseudocinerea associated with gray mould disease in peonies (Paeonia lactiflora Pall.) in Southern Chile.

BACKGROUND: In Chile, the peony is the most important ornamental flower exported from the country. Gray mould is a phytopathological problem of this crop. This disease is caused by Botrytis cinerea and Botrytis paeoniae. AIMS: We carried out the first survey of Botrytis species associated with peony gray mould in Southern Chile to estimate the diversity of these pathogens. METHODS: Diseased peony leaves were collected from seven locations in Southern Chile covering a distance of 300km. The Botrytis isolates obtained were studied by morphological and molecular methods. Finally, a PCR assay using primers based on the necrosis and ethylene-inducing protein gene (nep1) was used to specifically identify B. paeoniae. RESULTS: Seventeen isolates belonging to Botrytis genus were obtained, and all of them were pathogenic to peonies when inoculated in plants grown in a greenhouse. Morphological analyses showed that four isolates shared common characteristics, which distinguish them from the rest. Homology and phylogenetic analysis of G3PDH, as well as determination of the Bc-hch allele, allowed us to identify 12 isolates as B. cinerea, 4 as B. paeoniae and one isolate as Botrytis pseudocinerea. The PCR assay was found to be specific to B. paeoniae, amplifying a single band of 470bp. CONCLUSIONS: Three Botrytis species involved in peony gray mould disease are present in Chile. This is the first time that both B. paeoniae and B. pseudocinerea have been reported to be present in the country and also that they affect peonies. Finally, to our knowledge, the PCR based method herein described is the first of its kind to be used to identify B. paeoniae.