Sensitivity of Botrytis cinerea Isolates Complex to Plant Extracts.

New agricultural strategies aim to reduce the use of pesticides due to their damage to the environment and humans, and the caused resistance to pathogens. Therefore, alternative sources of antifungal compounds from plants are under investigation lately. Extracts from plants have a wide composition of chemical compounds which may complicate the development of pathogen resistance. Botrytis cinerea, causing grey mould, is an important horticultural and ornamental pathogen, responsible for the relevant yield and quality losses. B. cinerea isolated from a different plant host may differ in the sensitivity to antifungal substances from plants. Assessing the importance of research covering a wide range of pathogens for the rapid development of biopesticides, this study aims to determine the sensitivity of the B. cinerea isolate complex (10 strains) to plant extracts, describe morphological changes caused by the extract treatment, and detect differences between the sensitivity of different plant host isolates. The results showed the highest sensitivity of the B. cinerea isolates complex to cinnamon extract, and the lowest to laurel extract. In contrast, laurel extract caused the most changes of morphological attributes in the isolates. Five B. cinerea isolates from plant hosts of raspberry, cabbage, apple, bell pepper, and rose were grouped statistically according to their sensitivity to laurel extract. Meanwhile, the bell pepper isolate separated from the isolate complex based on its sensitivity to clove extract, and the strawberry and apple isolates based on their sensitivity to cinnamon extract.

Incidence, Distribution, and Pathogenicity of Fungi Causing Root Rot in Idaho Long-Term Sugar Beet Storage Piles.

Fungal rots in sugar beet roots held in long-term storage can lead to considerable sucrose loss but the incidence and distribution of fungal rots inside sugar beet piles and pathogenicity for some species is poorly understood. Thus, Idaho sugar beet held in five outdoor and two indoor piles in 2014 and 2015 were investigated. The root surface area covered by fungal growth and discolored and healthy tissue were assessed in nine 1-m2 areas per pile using a stratified random sampling design. Pathogenicity was evaluated indoors via plug inoculation in 2015 and 2016. Botrytis cinerea covered more root surface area inside indoor piles (6 to 22%) than outdoor piles (0 to 3%) (P < 0.0001). No trends were evident for the Athelia-like sp. (0 to 15%) and Penicillium-type spp. (0 to 8%). Penicillium-type isolates comprised the following species: 60% Penicillium expansum, 34% P. cellarum, 3% P. polonicum, and 3% Talaromyces rugulosus. Trace levels (<1% of root surface) of other fungi, including Cladosporium and Fusarium spp., were evident on roots and in isolations. Based on sample location in a pile, there were no trends or differences; however, two outdoor piles (OVP1 and OVP2) had more healthy tissue (90 to 96%) than other piles (28 to 80%) (P < 0.0001). When the pathogenicity tests were analyzed by species, all were significantly different from each other (P < 0.0001), except for P. polonicum and P. expansum: B. cinerea (61 mm of rot), P. polonicum (36 mm), P. expansum (35 mm), P. cellarum (28 mm), Athelia-like sp. (21 mm), T. rugulosus (0 mm; not different from check), and noninoculated check (0 mm). The OVP1 and OVP2 piles had negligible fungal growth on roots after more than 120 days of storage under ambient conditions, which indicates that acceptable storage can be achieved over this time period through covering piles with tarps and cooling with ventilation pipe.

Talaromyces pinophilus strain AUN-1 as a novel mycoparasite of Botrytis cinerea, the pathogen of onion scape and umbel blights.

This study aimed to investigate the mycoparasitism of Botrytis cinerea, the pathogen of scape and umbel blights of onion seed crops, by endophytic Talaromyces pinophilus. The dual culture test showed that the antagonistic potentiality of T. pinophilus against B. cinerea depend on the mycoparasitism that was morphologically detected by the formation of mycelial overgrowth. Moreover, the light micrograph of the mycelia at the contact zone exhibited that the hyphae of T. pinophilus penetrated and grew intracellularly inside the hyphae of B. cinerea. A more illustrative figure of the establishment of coiled hyphae as well as the conformation of the penetration process was assayed by SEM and TEM analyses. SEM micrograph revealed that the hyphae of T. pinophilus grew along hyphae of B. cinerea, attached, coiled around the host hypha and generated pseudoappressorium. A clear disintegration of cell wall of the host hypha was observed at the penetration site. The micrographs of TEM exhibited the ability of T. pinophilus to produce pseudoappressorium, penetrate and then entere a hypha of B. cinerea causing distinct cytoplasmic disorganization. High activities of cell wall degrading enzymes (chitinase, lipase and protease) involved in the mycoparasitism were evaluated by the endophytic T. pinophilus. In conclusion, this study demonstrated that the endophytic T. pinophilus may be a promising biocontrol agent against phytopathogenic fungi instead of chemical fungicides.

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.

Identification and Characterization of Botrytis Blossom Blight of Japanese Plums Caused by Botrytis cinerea and B. prunorum sp. nov. in Chile.

Blossom blight is a destructive disease of plums (Prunus salicina) when humid and temperate weather conditions occur in Chile. Disease incidence ranging from 4 to 53% has been observed. Symptoms include light brown petal necrosis, starting as light brown mottles or V-shaped necrosis at the margins of the petals, progressing to the stamen and pistils. In this study, the etiology of blossom blight of plums was determined. High- and low-sporulating isolates of Botrytis were obtained consistently from blighted blossoms and apparently healthy flowers of plums. Based on colony morphology, conidial production and molecular phylogenetic analysis, these high- and low-sporulating isolates were identified as B. cinerea and B. prunorum sp. nov., respectively. Phylogenetic analysis of the genes glyceraldehyde 3-phosphate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60), and DNA-dependent RNA polymerase subunit II (RPB2) grouped B. prunorum isolates in a single cluster, distantly from B. cinerea and other Botrytis species. The phylogenetic analysis of necrosis and ethylene-inducing protein (NEP1 and NEP2) genes corroborated these results. Analysis of the internal transcribed spacer region and large-subunit (26S) ribosomal DNA and detection of Boty and Flipper transposable elements, were not useful to differentiate between these Botrytis species. Both species were pathogenic on plum flowers and the fruit of plums, apples, and kiwifruits. However, B. prunorum was less virulent than B. cinerea. These pathogens were re-isolated from inoculated and diseased tissues; thus, Koch's postulates were fulfilled, confirming its role in blossom blight of plums. B. cinerea was predominant, suggesting that B. prunorum may play a secondary role in the epidemiology of blossom blight in plums in Chile. This study clearly demonstrated that the etiology of blossom blight of plums is caused by B. cinerea and B. prunorum, which constitute a species complex living in sympatry on plums and possibly on other stone fruit trees.

Fungal Endophyte Diversity and Bioactivity in the Indian Medicinal Plant Ocimum sanctum Linn.

Endophytic mycopopulation isolated from India's Queen of herbs Tulsi (Ocimum sanctum) were explored and investigated for their diversity and antiphytopathogenic activity against widespread plant pathogens Botrytis cinerea, Sclerotinia sclerotiorum, Rhizoctonia solani and Fusarium oxysporum. 90 fungal isolates, representing 17 genera were recovered from 313 disease-free and surface sterilised plant segments (leaf and stem tissues) from three different geographic locations (Delhi, Hyderabad and Mukteshwar) during distinct sampling times in consequent years 2010 and 2011 in India. Fungal endophytes were subjected to molecular identification based on rDNA ITS sequence analysis. Plant pathogens such as F. verticillioides, B. maydis, C. coarctatum, R. bataticola, Hypoxylon sp., Diaporthe phaseolorum, Alternaria tenuissima and A. alternata have occurred as endophyte only during second sampling (second sampling in 2011) in the present study. Bi-plot generated by principal component analysis suggested tissue specificity of certain fungal endophytes. Dendrogram revealed species abundance as a function of mean temperature of the location at the time of sampling. Shannon diversity in the first collection is highest in Hyderabad leaf tissues (H' = 1.907) whereas in second collection it was highest from leaf tissues of Delhi (H' = 1.846). Mukteshwar (altitude: 7500 feet) reported least isolation rate in second collection. Nearly 23% of the total fungal isolates were considered as potent biocontrol agent. Hexane extract of M. phaseolina recovered from Hyderabad in first collection demonstrated highest activity against S. sclerotiorum with IC50 value of 0.38 mg/ml. Additionally, its components 2H-pyran-2-one, 5,6-dihydro-6-pentyl and palmitic acid, methyl ester as reported by GC-MS Chromatogram upon evaluation for their antiphytopathogenic activity exhibited IC50 value of 1.002 and 0.662 against respectively S. sclerotiorum indicating their significant role in antiphytopathogenic activity of hexane extract. The production of 2H-pyran-2-one, 5,6-dihydro-6-pentyl from M. phaseolina, an endophytic fungus is being reported for the first time.

Avaliação do extrato etanólico de Ottonia martiana Miq. para o controle de duas doenças florestais / Evaluation of Ottonia martiana Miq. alcoholic extract to control two forest diseases

Metabólitos secundários presentes em plantas medicinais apresentam várias propriedades biológicas incluindo a atividade antifúngica. Esse estudo avaliou o potencial antifúngico da planta medicinal Ottonia martiana no controle da pinta-preta em erva-mate (Ilex paraguariensis) e do mofo-cinzento em eucalipto (Eucalyptus dunnii). Extrato etanólico (EBEtOH) dos órgãos totais (raízes, caules, folhas e frutos) foi preparado e testado na concentração de 1000 μg mL-1 contra os patógenos Cylindrocladium spathulatum (pinta-preta) e Botrytis cinerea (mofo-cinzento). Bioensaios in vitro (germinação de esporos e bioautografia direta) e in vivo (teste de patogenicidade em mudas) mostraram que o EBEtOH reduziu o crescimento micelial dos patógenos testados e a germinação dos esporos de C. spathulatum e estimulou a germinação de esporos de B. cinerea. O teste de patogenicidade mostrou que o controle da pinta-preta em erva-mate e do mofo cinzento em eucalipto não é viável usando-se a concentração testada de EBEtOH de O. martiana. Na bioautografia direta, foram detectadas zonas de inibição de crescimento micelial dos fungos e que foram relacionadas com a presença de piperovatina.

Secondary metabolites from medicinal plants have several biological properties, including antifungal activity. This study evaluated the antifungal potential of the medicinal plant Ottonia martiana to control maté leaf spot (Ilex paraguariensis) and eucalypt gray mould (Eucalyptus dunnii). Ethanol extract (EBEtOH) of the total parts (roots, stems, leaves and fruits) was prepared at the concentration of 1000 μg mL-1 and tested against Cylindrocladium spathulatum (maté leaf spot) and Botrytis cinerea (eucalypt gray mould). In vitro bioassays (spore germination and direct bioautography) and in vivo bioassays (pathogenicity test in seedlings) showed that EBEtOH reduced the mycelial growth of the tested pathogens and the germination of C. spathulatum spores and stimulated the germination of B. cinerea spores. The pathogenicity test showed that the control of maté leaf spot and eucalypt gray mould is not viable using the tested concentration of O. martiana EBEtOH. Zones of mycelial growth inhibition were detected in direct bioautography and were related to the presence of piperovatine.

[Effect of PEG stress on plantlets of Chrysanthemum morifolium induced by endophytic botrytis sp. (C1) and Chaetomium globosum (C4)].

The effect of the endophytic fungi Botrytis sp. (C1) or Chaetomium globosum (C4) on the drought resistance of Chrysanthemum morifolium was studied. Ch. morifolium plantlets were inoculated with C1, C4 and cultured in the pots for 60 days, then the plantlets were stressed by 0%, 10%, 20%, 30%, 40% PEG6000 respectively in order to simulate different drought conditions. Biomass, the activities of SOD, POD, PAL, the contents of MDA and soluble protein of each group were determined. The results showed that endophytic fungi groups grew better than the control (without inoculation endophytic fungi). With the increasing of the concentration of PEG6000, the biomass of Ch. morifolium of each groups decreased, while the biomass of fungi groups was significantly higher than that of control, moreover C4 group higher than C1 group. With the concentration of PEG increasing, the content of MDA of each group increased too, while POD activity and soluble protein content of all treatments increased at first and then decreased. SOD activity and PAL activity of the control were increased with the increase of PEG concentration, but SOD activity of the two fungi groups were stable. After been stressed by different concentrations of PEG, MDA content of two fungi groups were always lower than the control, while SOD activity, POD activity, PAL activity and soluble protein content were higher. In conclusion, endophytic fungi can increase the drought resistance of Ch. morifolium.