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1.
Plant Dis ; 2021 Jan 04.
Article in English | MEDLINE | ID: mdl-33393362

ABSTRACT

In January and March 2019, an inspection of 11 commercial 'Hass' avocado orchards in mid-North and Tauranga (New Zealand) was conducted by NZ Avocado Growers Association Inc. (NZAGA) and the samples were sent to Plant Diagnostics Limited for investigation of a newly observed fruit staining symptom termed "tannin stain". Fruit symptoms consisted of areas of minute small spots which coalesced into areas of tear staining associated with water movement over the fruit's surface (Supplementary Fig. 1). Up to seven trees per orchard were sampled targeting symptomatic fruit with the aim of determining the cause of the problem. Fruit was surface disinfected for 4 minutes in 1% sodium hypochlorite solution and sections from lesions were plated on agar medium (prune extract agar) to isolate any plant pathogens. The predominant fungi isolated, represented species in the Colletotrichum acutatum, C. gloeosporioides, and C. boninense species complexes. Since the morphological characters within these complexes overlap (see Supplementary Fig. 2 for examples), the isolates were differentiated by amplification and sequencing of the glyceraldehyde-3-phosphate dehydrogenase (GPDH) gene and, where necessary, the calmodulin (CAL) gene and/or the Apn2-Mat1-2 intergenic spacer region (ApMat) locus (Weir et al., 2012; Rojas et al., 2010). The sequence analysis revealed eight Colletotrichum species comprising C. alienum, C. aotearoa, C. cigarro, C. fioriniae, C. fructicola, C. karstii, C. perseae, and C. siamense. This range included three species that have not previously been recorded in New Zealand: C. fructicola (Cf), C. perseae (Cp), and C. siamense (Cs). Colonies for all these three fungi were white to grey with salmon-coloured and black acervuli. Conidia were aseptate, hyaline, straight, cylindrical, with broadly rounded ends, forming on cylindrical conidiogenous cells. The respective GPDH, CAL, and/or ApMat sequences of the Cf, Cp, and Cs isolates were identical to reference sequences of representative isolates in GenBank (e.g. ApMat: Cf - KX620181, Cp - KX620177, Cs - KP703788). An isolate for each species is stored in the International Collection of Microorganisms from Plants (Cf - ICMP22409, Cp - ICMP22431, Cs - ICMP22411) and sequences are deposited in GenBank (accession numbers MT522858-MT522865). Pathogenicity of each of the newly recorded species was confirmed on freshly picked 'Hass' avocado fruit. After surface disinfection with 1% sodium hypochlorite solution for 5 minutes, fruit was triple washed with sterile water and air dried. Five fruits per species were pin-pricked and inoculated with 10µL of conidial suspension (7 x 106 to 1 x 107 conidia/mL) prepared with sterile water containing Tween 20 (1µL/mL H2O) from 6-day-old cultures grown on PDA. Control fruit was pin-pricked and mock-inoculated with sterile water containing Tween 20 (1µL/mL H2O). All fruit was incubated in moist chambers at 25°C for 7 days. The three Colletotrichum species produced anthracnose symptoms on inoculated fruit whereas no symptoms were observed on control fruit (Supplementary Fig. 3). Each one of the species was successfully re-isolated from symptomatic tissue and identified using the methods described above, fulfilling Koch's postulates. While Cf and Cs have been reported from several hosts and countries to date (Weir et al. 2012), Cp has only been found from avocado in Israel (Sharma et al. 2017) and grape in Japan (Yokosawa et al. 2020). Although a number of species from the C. gloeosporioides species complex, i.e. C. alienum, C. aotearoa, C. cigarro, and C. gloeosporioides have been previously associated with avocado diseases in New Zealand, the detections of Cf, Cp, and Cs represent first records. In this study, eight Colletotrichum species were associated with the "tannin stain" fruit symptoms in New Zealand avocado orchards. The individual contribution of the newly recorded pathogens Cf, Cp, and Cs to the observed disease symptoms was not determined, but their detection highlights the importance of sequence-based identification of Colletotrichum species, as morphology is insufficiently robust to separate cryptic species. Accurate identification of pathogens provides knowledge of species biodiversity that may be useful in biosecurity decision making. Since it has been reported that fungicide treatment efficiencies differ for some closely related Colletotrichum species on grape (Yokosawa et al. 2020), accurate identification might also contribute to establishing effective management strategies.

2.
J Basic Microbiol ; 57(5): 419-427, 2017 May.
Article in English | MEDLINE | ID: mdl-28211948

ABSTRACT

In this study we examined the influence of the ambient pH during morphogenesis on conidial yield of Trichoderma sp. "atroviride B" LU132 and T. hamatum LU593 and storage at low temperatures. The ambient pH of the growth media had a dramatic influence on the level of Trichoderma conidiation and this was dependent on the strain and growth media. On malt-extract agar, LU593 yield decreased with increasing pH (3-6), whereas yield increased with increasing pH for LU132. During solid substrate production the reverse was true for LU132 whereby yield decreased with increasing pH. The germination potential of the conidia decreased significantly over time in cold storage and the rate of decline was a factor of the strain, pH during morphogenesis, growth media, and storage temperature.


Subject(s)
Trichoderma/physiology , Cold Temperature , Culture Media , Germination , Hydrogen-Ion Concentration , Morphogenesis , Spores, Fungal/growth & development , Spores, Fungal/physiology , Time Factors , Trichoderma/growth & development
3.
Front Plant Sci ; 8: 102, 2017.
Article in English | MEDLINE | ID: mdl-28232840

ABSTRACT

Trichoderma species are soil-borne filamentous fungi widely utilized for their many plant health benefits, such as conferring improved growth, disease resistance and abiotic stress tolerance to their hosts. Many Trichoderma species are able to produce the auxin phytohormone indole-3-acetic acid (IAA), and its production has been suggested to promote root growth. Here we show that the production of IAA is strain dependent and diverse external stimuli are associated with its production. In in vitro assays, Arabidopsis primary root length was negatively affected by the interaction with some Trichoderma strains. In soil experiments, a continuum effect on plant growth was shown and this was also strain dependent. In plate assays, some strains of Trichoderma spp. inhibited the expression of the auxin reporter gene DR5 in Arabidopsis primary roots but not secondary roots. When Trichoderma spp. and A. thaliana were physically separated, enhancement of both shoot and root biomass, increased root production and chlorophyll content were observed, which strongly suggested that volatile production by the fungus influenced the parameters analyzed. Trichoderma strains T. virens Gv29.8, T. atroviride IMI206040, T. sp. "atroviride B" LU132, and T. asperellum LU1370 were demonstrated to promote plant growth through volatile production. However, contrasting differences were observed with LU1370 which had a negative effect on plant growth in soil but a positive effect in plate assays. Altogether our results suggest that the mechanisms and molecules involved in plant growth promotion by Trichoderma spp. are multivariable and are affected by the environmental conditions.

5.
Methods Mol Biol ; 1477: 23-35, 2016.
Article in English | MEDLINE | ID: mdl-27565489

ABSTRACT

Members of the genus Trichoderma comprise the majority of commercial fungal biocontrol agents of plant diseases. As such, there is a wealth of information available on the analysis of their biocontrol potential and the mechanisms behind their superior abilities. This chapter aims to summarize the most common methods utilized within a Trichoderma biocontrol program for assessing the biological properties of individual strains.


Subject(s)
Biological Control Agents , Trichoderma/physiology , Antibiosis , Cell Culture Techniques , Metabolome , Plant Diseases/microbiology , Plant Roots/microbiology , Rhizosphere , Trichoderma/classification , Trichoderma/isolation & purification , Triticum/microbiology , Volatile Organic Compounds/metabolism , Zea mays/microbiology
6.
Microbiology (Reading) ; 161(11): 2110-26, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26341342

ABSTRACT

Several members of the genus Trichoderma are biocontrol agents of soil-borne fungal plant pathogens. The effectiveness of biocontrol agents depends heavily on how they perform in the complex field environment. Therefore, the ability to monitor and track Trichoderma within the environment is essential to understanding biocontrol efficacy. The objectives of this work were to: (a) identify key genes involved in Trichoderma sp. 'atroviride type B' morphogenesis; (b) develop a robust RNA isolation method from soil; and (c) develop molecular marker assays for characterizing morphogenesis whilst in the soil environment. Four cDNA libraries corresponding to conidia, germination, vegetative growth and conidiogenesis were created, and the genes identified by sequencing. Stage specificity of the different genes was confirmed by either Northern blot or quantitative reverse-transcriptase PCR (qRT-PCR) analysis using RNA from the four stages. con10, a conidial-specific gene, was observed in conidia, as well as one gene also involved in subsequent stages of germination (L-lactate/malate dehydrogenase encoding gene). The germination stage revealed high expression rates of genes involved in amino acid and protein biosynthesis, while in the vegetative-growth stage, genes involved in differentiation, including the mitogen-activated protein kinase kinase similar to Kpp7 from Ustilago maydis and the orthologue to stuA from Aspergillus nidulans, were preferentially expressed. Genes involved in cell-wall synthesis were expressed during conidiogenesis. We standardized total RNA isolation from Trichoderma sp. 'atroviride type B' growing in soil and then examined the expression profiles of selected genes using qRT-PCR. The results suggested that the relative expression patterns were cyclic and not accumulative.


Subject(s)
Genetic Markers , RNA, Fungal/genetics , Soil Microbiology , Trichoderma/growth & development , Blotting, Northern , Gene Expression Profiling/standards , Gene Library , Molecular Sequence Data , RNA, Fungal/isolation & purification , Real-Time Polymerase Chain Reaction/standards , Sequence Analysis, DNA , Trichoderma/cytology , Trichoderma/genetics
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