Taxonomy, phylogeny and characterization of selected pathogenesis factors of microfungi associated with strawberry and grapevine

Name: Janith Vishvakeerthi Sri Aluthmuhandiram

Address: 333 Moo1, Thasud, Muang, Chiang Rai 57100

Organization : Mae Fah Luang University

Email : library@mfu.ac.th

keyword: Ascomycota

MeSH: Fungi

Classification :.DDC: e-thesis

; Hyphomycetes

MeSH: Phylogeny

MeSH: Biology -- Classification -- 59923

MeSH: Grapes

MeSH: Strawberries -- 156380

LCSH: Fungi

LCSH: Phylogeny

LCSH: Biology -- Classification -- 59923

LCSH: Grapes

LCSH: Strawberries -- 156380

Abstract: Strawberry (Fragaria × ananassa) and grapevine (Vitis vinifera) are major economic crops in the global fruit industry and in China. In this study, the taxonomy and phylogeny of microfungi associated with these crops were investigated, and the pathogenesis of selected fungal species was evaluated. Specimens of strawberry and grapevine were collected from agriculture fields in China and Thailand. Fungal identification was mainly conducted based on morphology and multi-gene phylogeny. The impact of temperature variation on the production of phytotoxic secondary metabolites by Lasiodiplodia theobromae was evaluated. Additionally, the role of a putative NmrA-like protein, Lws1, in Lasiodiplodia theobromae was investigated through gene silencing and overexpression experiments to assess its pathogenic function. The findings of this dissertation summarized microfungi associated with decaying and dead strawberry flowers identified based on morphology and phylogeny. Samples were collected from commercial fields in northern Thailand. Six microfungal taxa from the families Cladosporiaceae, Nectriaceae, and Pleosporaceae, associated with decaying or diseased flower parts (anthers, apocarpous gynoecium, petals, and sepals) were identified. This study introduces Cladosporium fragariae and Fusarium petalifragariae as new species based on morphology and multi-gene phylogeny. Cladosporium subuliforme, Curvularia geniculata, and Fusarium sulawesiense are reported for the first time as being associated with Fragaria × ananassa. Additionally, Cladosporium maltirimosum is reported for the first time in Thailand from strawberry, as a new host record. The fungal groups identified in this research can cause pre-harvest flower diseases in strawberry fields. Additionally, the reported fungal taxa may function as secondary invaders, acting as saprotrophs on necrotic plant tissue. Pathogenicity tests are essential to determine whether these fungi exhibit necrotrophic or saprotrophic lifestyles and should be conducted under field conditions in future. Further sampling, identification, and experimental confirmation are required to validate these findings. Fusarium is a pathogenic genus associated with root diseases in strawberries. Symptomatic strawberry plants were collected, exhibiting wilted foliage and dried, withered leaves. Stunted plant growth and reduced fruit production were also observed in these specimens. Fusarium species associated with the roots were isolated using the tissue isolation technique. Morphological identification was conducted alongside multi-locus phylogeny based on combined multiple sequence alignments (MSA) of RNA polymerase largest subunit (RPB1), RNA polymerase second largest subunit (RPB2), and translation elongation factor 1-alpha (TEF1-α) sequences. Morphological analysis revealed three new species: Fusarium fragariae sp. nov., Fusarium miyunense sp. nov., and Fusarium yanqingense sp. nov. Seven new host records were established for Fusarium annulatum, F. cili, F. graminearum, F. laceratum, F. nirenbergiae, F. verticillioides, and Neocosmospora pisi. The necessity of field experiments to evaluate the pathogenicity of the above isolates was highlighted. Therefore, the term “associated with” was used to describe the identified isolates. In another investigation, dead strawberry leaf specimens infected with hoar-frost fungi were collected in Chiang Rai, Thailand, and identified using phylogenetic analysis and morphological identification. A polyphasic approach was employed to introduce the isolate as Botryosporium longibrachiatum. In this study, the taxonomic classification of Botryosporium was revised to Catabotryales (Diaporthomycetidae). A new family, Botryosporiaceae, was proposed based on distinct asexual morphology and multi-gene phylogeny. This research is significant as it compiles morphological findings related to Botryosporium since 1998. The study represents the first comprehensive multi-gene phylogenetic analysis for the genus, resolving the placement of the genus. Botryosphaeriaceae species were associated with grapevine trunk diseases. Lasiodiplodia theobromae was identified as a significant grapevine pathogen. In this study, the effects of temperature variation on phytotoxin-induced virulence in Lasiodiplodia theobromae were evaluated. The fungal strain was grown in Czapek-Dox liquid medium at four temperatures for 14 days. Culture filtrates were screened for three selected phytotoxins using UPLC MS/MS techniques. The phytotoxicity of the crude culture filtrates was tested on detached grapevine leaves. UPLC MS/MS analysis identified variations in 3-Indolinecarboxylic acid and jasmonic acid production with increasing temperature. No lasiodiplodin was detected during the analysis. Significant increases in phytotoxicity of crude culture filtrates were observed at higher temperatures. The most severe necrotic leaf lesions were observed with filtrates extracted at 35 °C. NmrA-like proteins were reported as important nitrogen metabolism regulators and virulence factors. However, their role in the woody plant pathogen Lasiodiplodia theobromae remained unclear. In the current study, a putative NmrA-like protein, Lws1, was identified in L. theobromae. Its pathogenic role was investigated via gene silencing and overexpression experiments. The effects of external carbon and nitrogen sources on Lws1 gene expression were evaluated using qRT-PCR assays. Molecular interactions between Lws1 and its target protein were analyzed using the yeast two-hybrid system. The results showed that Lws1 contained a canonical glycine-rich motif shared by SDR superfamily proteins. Lws1 functioned as a negative regulator during disease development. Transcription profiling revealed that external nitrogen and carbon sources affected Lws1 transcription. Interaction analyses demonstrated that Lws1 interacted with a putative GATA family transcription factor, LtAreA. In conclusion, these results suggested that Lws1 was a critical regulator in nutrition metabolism. In conclusion, the present study provides significant insights into the taxonomy and pathogenicity of microfungi associated with strawberry and grapevine crops. The identification of new fungal species and host records, particularly within the genera Cladosporium, Fusarium, Curvularia, and Botryosporium, serves as a basis for further investigating their potential pathogenicity on strawberries. The findings regarding the phytotoxic effects of Lasiodiplodia theobromae and the regulatory function of the NmrA-like protein Lws1 further emphasize the importance of studying the effects of environmental factors, such as temperature, on fungal virulence, highlighting important implications for climate change and its potential to heighten fungal virulence. Furthermore, it is evident that nutrient levels play a crucial role in the function and expression of Lws1 in Lasiodiplodia theobromae on grapevine. Overall, the present research findings enhance our understanding of strawberry-associated microfungi. This study also emphasizes the effects of phytotoxic secondary metabolite production and the protein function of Lasiodiplodia theobromae under varying temperatures and nutrient levels, deepening our understanding of the pathogenesis of Lasiodiplodia theobromae on grapevines. These findings will lay the groundwork for future research focused on climate change, pathogenesis, and effective mitigation strategies to prevent significant damage to strawberries and grapevines. Future studies on pathogenicity assays and molecular analyses are essential to validate the present findings under expanded field conditions.

Mae Fah Luang University. Learning Resources and Educational Media Centre

Address: CHIANG RAI

Email: library@mfu.ac.th

Name: Thilini Chethana Kandawatte Wedaralalage

Role: Advisor

Created: 2024

Modified: 2024-12-17

Issued: 2024-12-17

วิทยานิพนธ์/Thesis

application/pdf

CallNumber: e-thesis

eng

DegreeName: Doctor of Philosophy

Level: Doctoral Degree

Descipline: Biosciences

Grantor: Mae Fah Luang University

©copyrights Mae Fah Luang University

ลำดับที่.	ชื่อแฟ้มข้อมูล	ขนาดแฟ้มข้อมูล	จำนวนเข้าถึง	วัน-เวลาเข้าถึงล่าสุด
1	138824.pdf	16.85 MB	3	2026-02-19 14:49:05

ใช้เวลา

0.037476 วินาที