Production of Phytotoxic Metabolites by Botryosphaeriaceae in Naturally Infected and Artificially Inoculated Grapevines

By Pierluigi Reveglia, Antonio Evidente, Alessio Cimmino, Marco Masi, Jennifer Millera Niem,Regina  Billones-Baaijens, Sandra Savocchia

Grapevine trunk diseases (GTDs) represent a serious threat to vineyards worldwide causing substantial economic and yield losses. To date, no curative methods are available and the connection between pathogen and symptom expression is not completely understood [¹]. Among the GTDs, Botryosphaeria dieback (BD) is considered a serious problem worldwide causing cankers, dieback and eventually death of vines. At least 27 species of Botryosphaeriaceae are associated with BD, although only 11 species have been reported in Australian vineyards to date [¹]. In Europe, the disease is also associated with foliar symptoms, although these symptoms have not been reported in Australian vineyards [²]. The expression of foliar symptoms is usually associated with phytotoxic metabolites (PMs) produced by the pathogen that are translocated into the leaves [³]. Considering this background and the absence of foliar symptoms in Australian vineyards, the following questions arose: (i) do the Botryosphaeriaceae spp. that infect grapevines in Australia produce PMs in vitro ?; (ii) are the PMs produced in vitro similarly produced in planta and get translocated into the host plant during pathogen attack?; and (iii) are the PMs involved in symptom expression?

Addressing these questions was the focus of my research project and served as the main objectives of my PhD at Charles Sturt University, Wagga Wagga, Australia and University of Naples Federico II, Naples, Italy. Prior to these investigations, limited information was available on the PMs produced by Australian isolates of Botryosphaeriaceae spp. 

In 2016, I began studying the ability of the most prevalent Australian Botryosphaeriaceae to produce PMs in vitro. My results revealed all BD pathogens produced PMs in vitro. Some of the purified PMs (Figure 1) were isolated and characterized for the first time and were demonstrated to have phytotoxic effect in detached leaves [^4–8].

The results presented an affirmative answer to the first question, which allowed me to move forward to address the succeeding questions. I developed a more complex experiment that aimed to investigate the production of PMs by Botryosphaeriaceae pathogens in naturally and artificially infected grapevines, and their likelihood of translocation in woody tissue. A multifaceted approach using a combination of molecular (quantitative PCR) and analytical chemistry (LC/MS-MS) techniques was used for these investigations (Figure 2).

Presumably, the most intriguing finding of this investigation was that (R)-mellein (1) was detected onlyin non-necrotic wood from an infected vine [⁹]. Thissuggests that this PM was not translocated (at least in these experimental conditions) throughout the wood which is the general hypothesis for PMs involved in GTDs [¹⁰]. These data highlight that the mere presence of PMs in the wood may not be enough to result in foliar symptoms on grapevines with BD. The development of symptoms could be more complex than previously thought and they could arise from the interaction between biotic and abiotic stress (water stress, drought, heat stress etc) [¹¹]. Different stress factors can weaken the plant and these may predispose the grapevine to foliar symptoms.

Moreover, it is very likely that a diseased grapevine shows differences in its metabolome compared to a healthy grapevine. Thus, metabolomic studies might disclose important insights on what happens to a grapevine when it is exposed to different abiotic and biotic stress conditions. Metabolomics is the latest of the omics sciences and its relevance in plant science, including GTDs investigation, has increased in the last decades [^12,13].

Investigations using complex mixtures, such as wood extracts obtained under different stress conditions, could result in the identification of unique chemical fingerprints that may be related to specific cellular processes, such as defence mechanisms or stress responses of the plant. The analytical chemistry method developed during the above described study has all the characteristics for being suitable for metabolomics research, and may shed light on the metabolic changes that occur in a grapevine infected by Botryosphaeriaceae pathogens. 

In conclusion, more comprehensive field studies on the influence of climatic conditions on foliar symptoms associated with GTD, including the role of PMs produced in planta, are fundamental for elucidating the relationship between fungal PMs and physiological changes in the vine which result in the expression of foliar symptoms. Indeed, multi-disciplinary research is required to contribute to the global understanding of the pathogen life cycle and disease development in vineyards, promoting the development of more specific and environmentally friendly control method for grapevine diseases.

Read more at: https://www.mdpi.com/2223-7747/10/4/802

Pierluigi Reveglia is a Post Doctoral Researcher at the University of Foggia, Italy. The main focus of his current research is the study of lipids factors that could be associated with the hyperphosphorylation of the Tyr-682 residue in the APP in Alzheimer Disease. Dr Pierluigi Reveglia completed a Co-tutele PhD course, between Charles Sturt University (Wagga Wagga, Australia) and University of Naples Federico II (Naples, Italy), with a Thesis entitled ” Isolation and characterisation of phytotoxins produced by Botryosphaeriaceae and their role in grapevine trunk diseases”. Pierluigi was awarded the Higher Degree by Research University Medal in 2019. Before moving to Australia to study a co-tutele PhD, Pierluigi has a research fellowship in isolation and chemical and biological characterization of phytotoxins by pathogenic fungi of legumes at the Department of Chemical Sciences of the University of Naples “Federico II”. In 2015 he completed the Master degree in Chemical Sciences with full mark and honours at the University of Naples “Federico II”. During his studies, he worked mainly in organic synthesis, dealing with the synthesis of C-glycosides with possible biological activity. In 2012 he completed the Bachelor Degree in Chemistry with full mark and honours. In 2010 It was awarded an LLP-Erasmus (European exchange program) scholarship. He studied for nine months at the Universidad de Alcala, Alcala de Henares, Spain. During that period, he focused his study on biochemistry and researched the field of organic electrosynthesis.

Antonio Evidente. He became researcher in organic chemistry in 1980 and the moved as associated professor of Organic Chemistry at the University of Basilicata, Potenza, Italy. In 1989 he returned at the University of Naples “Federico II”. In 2000 he became full professor of organic chemistry and in Prof. Evidente have over 45 years experience in the organic chemistry of natural substances. Prof. Evidente is leader of several national and international research projects. Prof. Evidente coordinates national and international research projects (Regional and EU project) and actively participate to several EU COST Actions.  He coordinates bilateral project with University of Naples Federico II and some Universities in USA, Australia e South Africa. He was invited for a talk to four congresses of the American Chemical Society since 2006 to 2014. He is member of the Editorial board of Journal of Agriculture and Food Chemistry, Chirality, Biomolecules and Phytopathologia Mediterranea. Award “Medaglia Quilico” 2014, assigned for its researches in the field of chemistry of bioactive metabolites from plants and fungi, from the Organic Chemistry Division of Italian Chemical Society, research field in which he is considered a ‘world authority’.

Alessio Cimmino is Associate Professor of organic chemistry since October 1, 2018 at the Department of Chemical Sciences, University of Naples Federico II. He has acquired over 15 years of experience mainly in organic chemistry, spectroscopy and hemisynthesis of bioactive metabolites (phytotoxins, plant growth regulators, antibiotics, mycotoxins, fungicides, phytoalexins, elicitors, herbicides, proteins and polysaccharides) produced by phytopathogenic and antagonistic fungi and bacteria and plants. In particular, the stereostructural determination and hemisynthesis of bioactive metabolites and studies on structure-activity relationships and on their action mode have been carried out by using advanced spectroscopic and chemical methods. Dr. Cimmino has established national and international collaboration with Research Centres and Universities and has participated to several scientific international (European) and national (Italian and Spanish) research projects admitted at financing on the basis of competitive calls that provide peer review. He has been supervisor of MSc and PhD students and speakers at national and international conferences.  

Marco Masi is a fixed term researcher from December 2018 at Department of Chemical Sciences of University of Naples Federico II. Hi is also working as principal investigator on a project entitled “Buffelgrass biocontrol in the Sonoran Desert with foliar pathogen phytotoxic metabolites” in collaboration with Biotechnology and Biological Control Agency, BBCA, Rome, Italy and US Forest Service Rocky Mountain Research Station, Shrub Sciences Laboratory, Provo, USA. He has acquired over 12 years of experience mainly in organic chemistry, spectroscopy and hemisynthesis of bioactive metabolites (phytotoxins, plant growth regulators, antibiotics, mycotoxins, fungicides, herbicides, proteins and polysaccharides) produced by fungi, bacteria and plants. The structural determination of the isolated bioactive metabolites allowed him to deepen the knowledge of spectroscopic techniques, including 1D and 2D 1H and 13C NMR, and HRESI, APCI and EI mass spectrometry. Several studies carried out on the mode of action and on structure-activity relationship give him the possibility to improve his experience in the organic chemistry reactions.He is co-author of 144 scientific publications on international journals and more than 25 communications at national and international congresses. He has been supervisor of MSc and PhD students and speaker at national and international conferences.

Jennife Millera Niem is a University Researcher I at the Mycology section of University of Philippines Los Banos studying the diversity of macrofungi in Philippine forests and small islands. She completed her BSc degree in Agriculture majoring in Plant Pathology from University of the Philippines Los Baños (UPLB) and her MSc degree in Plant Pathology from Washington State University, USA. Dr Niem completed her PhD course at  Charles Sturt University (Wagga Wagga, Australia) in 2021 with a Thesis entitled ” Biological control of grapevine trunk diseases using bacterial endophytes from grapevines “.  Prior to that, she was employed for two years at the International Rice Research Institute (IRRI) as a mycotoxin specialist assessing the extent of mycotoxin contamination in rice in the Philippines and in Myanmar, evaluating mycotoxin detection techniques using various commercially available ELISA kits and immunochromatography-based methods and developing different mycotoxin reduction strategy.  She has also worked as University Research Associate at UPLB working on different research projects concerning fungal and bacterial diseases of agricultural crops before and after harvest. In a CDR-USAID funded collaborative project between UPLB and Volcani Center, Israel, she spent 2 years in Israel working on a research project comparing pectolytic and cellulolytic gene expressions in apple cultivars that differ in their susceptibility to Alternaria alternata, causal agent of apple core rot.

Regina “Reggie” Billones-Baaijens is currently employed as a Post Doctoral Research Fellow at the NWGIC. She is currently working on grapevine trunk disease management for vineyard longevity in diverse climates of Australia. The aim of the research project is to investigate the epidemiology along with development of efficient methods for pathogen detection, pruning wound management and control of these trunk disease pathogens in Australian vineyards. She completed her PhD in Plant Pathology at Lincoln University, New Zealand in 2011, a Masters degree in Applied Science at Massey University in 1999 and a Masters degree in Plant Pathology, minor in Molecular Biology at the University of the Philippines – Los Baños in 2005. Her PhD research focused on the role of nurseries in the spread of Botryosphaeria dieback in New Zealand vineyards. This disease is considered a serious problem of grapevines worldwide causing cankers, dieback and eventually death of vines. Prior to her employment as Post Doctoral Research fellow at the NWGIC, she worked as a Tutor in the Ecology Department at Lincoln University, New Zealand. This involved the delivery of lectures for undergraduate diploma courses and managing laboratory classes for Fungal Biology, Plant Protection and Molecular Biology undergraduate subjects. She also provided technical assistance to postgraduate students and contributed to the research programmes in the Plant Microbiology Group at Lincoln University.

Sandra Savocchia has a Bachelor of Agricultural Science (Honours) and a PhD from The University of Adelaide majoring in fungicide resistance and viticultural plant pathology. After completing her PhD she was employed at The University of Adelaide as a researcher in the area of grapevine root pest (phylloxera and root-knot nematode) resistance and was also involved in demonstrating and tutoring undergraduate students. She also worked for a private viticultural consultant in the Southern Vales region of South Australia. Sandra began lecturing at CSU in 2002 in viticulture and wine science and is currently involved in teaching both undergraduate and postgraduate students. She is passionate about her research and is involved in a number of plant pathology related projects including a national project studying the management of grapevine wood diseases. Sandra is currently Associate Dean, Graduate Studies for the Faculty of Science.

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