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3rd International Symposium on Weeds and Invasive Plants October 2-7, 2011 in Ascona, Switzerland New pathogens of Solanum elaeagnifolium investigated as possible biological control agents of the weed in Greece Javid Kashefi1,* and Anastasia L. Lagopodi2 ARS European Biological Control Laboratory, Thessaloniki, Greece 2 Plant Pathology Laboratory, School of Agriculture, Aristotle University, Thessaloniki, Greece 1 USDA ABSTRACT: Solanum elaeagnifolium (Silverleaf nightshade) is an alien invasive weed in Greece, parts of southern Europe and many North African countries with Mediterranean climate. Since its establishment in Greece in early 1900’s, it has become rapidly one the most dangerous and aggressive weeds in the country, especially in north and central parts. Climate change and the rise of temperature in the Mediterranean basin increase the possibility of spread and establishment of the weed in new regions, since it is extremely resistant to drought. Recently two new fungi were discovered attacking the weed in Greece and the goal is to evaluate the impact of their presence, and the possibility of using endemic pathogens for biological control of S. elaeagnifolium. The first fungus has been identified as Verticillum dahliae. However, V. dahliae is a serious pathogen and most of its strains are pathogenic to numerous hosts, so it is not appropriate as a biological control agent. On the other hand the presence of this fungus on a widespread weed is a threat to the cultivated plants, since diseased S. elaeagnifolium would facilitate the maintenance and proliferation of the fungus’s propagules in the soils. The second fungus is Erysiphe sp. Chances of Erysiphe sp. to be specific to S. elaeagnifolium are very high, since most powdery mildew pathogens are host specific, but this needs further justification. This is the first world record of both fungi attacking silverleaf nightshade. The use of endemic natural enemies of the invasive, alien weeds in Europe would bring the challenge of their control to a new level. KEYWORDS: Solanum elaeagnifolium, Verticillum dahliae, Erysiphe sp., biological control 1 INTRODUCTION Solanum elaeagnifolium is a perennial plant 20 cm to 1.2 m in height. The stems are covered with short spines, ranging from very few on some plants to very dense on others. Leaves and stems are covered with downy hairs that lie against and hide the surface, giving a silvery or greyish appearance. The leaves are up to 10 cm long and 0.5 to 2.5 cm wide, with shallowly waved edges. The flowers, appearing from April to August, have five petals united to form a star, ranging from blue to pale lavender or white; five yellow stamens and a pistil form a projecting centre. The plant produces yellow or orange berries that last all winter and may turn brown as they dry. According to recent studies, (MarieClaude Bon, personal communication), the origin of the Greek population is Texas, USA and probably it has been introduced into Greece by contaminated seeds around 1930. The epicenter of the weed in Greece is central Macedonia with the heaviest infestations. However, it has been reported from all the other regions of the country. Studies concerning the economic impacts of the weed in Greece have not been conducted yet. Control of the weed is extremely difficult and confounded by its ability to produce thousands of viable seeds from a single plant, Corresponding author address: Javid Kashefi, USDA ARS European Biological Control Laboratories, Thessaloniki, Greece; Tel.: +30 2310 473252; fax: +30 2310 473272; e-mail: [email protected] as well as a deep and well developed root system, which gives to the plant the ability to propagate vegetatively from creeping lateral roots, root fragments and rhizomes. The weeds’ resistance to drought and harsh environmental conditions is well known and documented. The most usual used methods of control are ploughing and the use of strong herbicides which brings short time relief but no permanent solution. Intensive mowing all year around can help to control smaller infestations in farms and gardens. Some of the classical biological control methods use natural enemies of the target organism in order to minimize its population to a tolerant level. The method has several advantages as compared to chemical control. Most of all it is environmentally friendly, sometimes it is the only control method available and can be more easily accepted by the public. Fungal pathogens over other plant pathogens are preferably used as biological control agents because they are easy to reproduce, easy to maintain and require no vectors for their application. A principle of biological control using fungi is that they should be host specific on the target host and should not cause diseases to other plants (Gnanamanikam et al., 2002). As S. elaeagnifolium is a relatively new species for Greece, there is no information about its natural pathogens in this country. The goal of this research is to investigate areas with dense popu- 3rd International Symposium on Weeds and Invasive Plants October 2-7, 2011 in Ascona, Switzerland lations of S. elaeagnifolium, report the occurrence of new fungal diseases, and further on to investigate the possibility that these pathogens are used as potential biological control agents of this weed. 2 MATERIALS AND METHODS 2.1 Area and time of research Field surveys were initiated in late summer 2009 in fields near Thermi and Triadi, areas in Thessaloniki prefecture northern Greece. Fields with S. elaeagnifolium were periodically visited from spring, a time when new plants growing from old rhizomes are blooming, until late summer-autumn, when the plant’s fruit is approaching maturity. 2.2 Occurrence of diseases, description of symptoms and identification of pathogens A high incidence of a disease with plants showing severe wilt symptoms was observed over large areas around Thermi, in late summer 2009. In some cases plants showed wilting symptoms on one side. Diseased plants were retarded in growth, dried out prematurely, and most of them produced few or no fruit. Examination of internal tissues revealed extensive dark discoloration of the vascular system. A fungus producing slowly growing white colonies, which eventually turned to black, was repeatedly isolated from the infected tissues. Microscopic examination of the colonies showed that the fungus produced spores on conidiophores characteristic for Verticillium dahliae Kleb. (Goud et al., 2003). Hyphae gave rise to microsclerotia which turned the colour of the colony white culture to black. The fungus was molecularly identified after amplification of the internal transcribed spacer 1 (ITS1) and ITS2 regions, including the ribosomal 5.8S RNA gene (White et al. 1990). During summer 2010 another disease was observed on silverleaf nightshade over large areas in Triadi. Leaves exhibited yellowish areas covered with a fine whitish mycelium, typical of a powdery mildew infection, and dried out prematurely. The mycelial masses expanded over the leaf, covering much of its surface, turning to whitish-grey. Infected plants died prematurely. Pin-point, black round structures were observed, scattered in the mycelial mass, when leaves started to dry out. Microscopic examination of the infected leaves showed numerous barrelshaped conidia. The tiny, dot-like black structures were cleistothecia that lacked characteristic appendages, and in late autumn contained numerous asci with ascospores. Based on the presence and morphology of cleistothecia and the number of asci the sexual stage of the fungus was recognized as an Erysiphe species, and the barrel-shaped conidia were considered as spores of its anamorph (Hanlin, 1990). 2.3. Pathogenicity tests and investigation of host range First, pathogenicity of V. dahliae isolated from S. elaeagnifolium was tested on the weed itself in order to fulfil Koch’s postulates. After proving pathogenicity on its original host, the fungus was used to inoculate paprika (Capsicum annuum) plants. Additionally, two virulent isolates of V. dahliae, one from eggplant, and another from olive tree were tested for pathogenicity on S. elaeagnifolium. Fungal inocula were prepared by growing each fungus in 250ml Czapek-Dox broth, in 500ml Erlenmeyer flasks, at 23oC, under continuous shaking, for 1 week. The cultures were filtered through 4 layers of cheesecloth, and the collected number of conidia was adjusted to a concentration of 106 spores/ml, by the use of a haematocytometer. S. elaeagnifolium seedlings grew in Jiffy peat pellets for 6 weeks prior to inoculation. The outer fine net of each Jiffy peat pellet was removed, so tiny wounds would be produced on the roots and the whole content of the Jiffy, containing the seedling, was dipped in V. dahliae inoculum for 10 min. Twenty S. elaeagnifolium seedlings were inoculated with each of the three V. dahliae isolates, and 20 seedlings dipped in deionized water served as control. Paprika seedlings grew in a nursery for 4 weeks prior to inoculation and were dipped with naked roots in V. dahliae inoculum. A slight trim of the paprika roots, in order to produce tiny wounds, took place prior to inoculation. Twenty plants were inoculated with V. dahliae isolated from S. elaeagnifolium, and another 20 plants dipped in de-ionized water served as control. The inoculated plants grew for 6 weeks in a greenhouse. They were visually examined for wilt symptoms, and the disease progress was followed for up to 6 weeks post inoculation. At the same time isolations from surface disinfected tissues were made, from root and stem of the inoculated plants, exhibiting severe wilt symptoms, in order to confirm the success of the artificial inoculations. The experiment was repeated twice. Pathogenicity of Erysiphe sp. isolated from S. elaeagnifolium was tested on the weed itself in order to fulfil Koch’s postulates. Release of ascospores and artificial inoculation by these spores was not successful. Inoculations by conidia were made by dusting the spores, with the aid of a fine brush over the leaves of wet, 8 weeks old S. elaeagnifolium seedlings, in pots. 3rd International Symposium on Weeds and Invasive Plants October 2-7, 2011 in Ascona, Switzerland The leaves were then closed in plastic bags for 2 days. Ten seedlings were artificially inoculated with conidia and another 10 seedlings with wet leaves, closed in plastic bags, for 2 days, were used as controls. Seedlings were examined for symptoms of powdery mildew for the following 12 days. 3 RESULTS AND DISCUSSION V. dahliae isolated from S. elaeagnifolium was proved to be extremely virulent on its original host. All the artificially inoculated seedlings of S. elaeagnifolium developed severe wilt symptoms and all of them died within 3 weeks post inoculation. The fungus was successfully re-isolated from stems and roots of all inoculated plants. The V. dahliae isolate from S. elaeagnifolium was also proved to be extremely virulent on paprika. A hundred percent of artificially inoculated paprika seedlings developed severe disease symptoms in 4 weeks post inoculation, and the fungus was successfully reisolated from stems and roots of all inoculated paprikas. At the same time, S. elaeagnifolium was successfully inoculated from two other V. dahliae isolates originating from eggplant and olive tree. All seedlings developed typical wilt symptoms 6 weeks post inoculation. The fungi were re-isolated from roots and stems from artificially inoculated S. elaeagnifolium. V. dahliae is a serious pathogen worldwide, causing great losses in many plant crops. It attacks many plant species causing wilt diseases. Strains of this fungus usually are not host specific. The fungus once established in a field is practically impossible to remove, since it can survive for years forming microsclerotia. At the same time it attacks several weeds, which by this way help the pathogen to spread and form more propagules in the soil. The results of the present work demonstrate that S. elaeagnifolium can be infected by V. dahliae strains isolated from common horticultural crops. At the same time the strain isolated from this weed can infect cultivated plants such as paprika. These findings indicate that S. elaeagnifolium exerts two serious threats for the cultivated plants in northern Greece. Not only it is a weed difficult to control, but at the same time it carries a very serious pathogen, V. dahliae and contributes to its maintenance in soil. For this reason it is extremely urgent that effective measures should be implemented for the control of this weed. To the best of our knowledge, this is the first world record of V. dahliae attacking Silverleaf nightshade. At present, possible other hosts of the strain isolated from S. elaeagnifolium are tested, including tomato, eggplant, cucumber, tobacco and olive tree. S. elaeagnifolium plants inoculated with Erysiphe sp. developed the disease symptoms 8 days after inoculation. Forty percent of inoculated leaves were infected and eventually dried out. A powdery mildew pathogen such as Erysiphe sp. could possibly act as a successful biological control agent due to its host specificity and usually high virulence. To the best of our knowledge, this is the first world record of Erysiphe sp. attacking silverleaf nightshade. At present the molecular identification of the fungus is taking place. However the work presented here can only be considered as a preliminary study. Pathogenicity of the fungus was confirmed in artificial inoculations, however possible other hosts must be checked for susceptibility, since in some cases, pathogenicity on more than one host has been reported for some powdery mildew pathogens (Smith 1969, Rector et al., 2005). Experiments should include other plants especially in the Solanaceae family such as tomato, tobacco, eggplant and paprika, and possibly on members of other families, depending on the species of the fungus. Conditions of artificial inoculations should be optimized, in order to develop a high disease incidence. Plant pathogens have successfully been used as biological control agents due to their host specificity (Charudatan, 2001). Erysiphe spp. have been suggested for biological control of weeds in the past (Ialongo, 1980). The use of endemic natural enemies of the invasive, alien weeds in Europe brings the challenge of their control to a new level in the beginning of 21st century Field surveys for new diseases of S. elaeagnifolium are at present continued. 4.3 References Charudattan, R. 2001. Biological control of weeds by means of plant pathogens: Significance for integrated weed management in modern agro-ecology. BioControl 46: 229260. Goud, J. C., Termorshuizen, A. J., & Gams, W. 2003. Morphology of Verticillium dahliae and V. tricorpus on semi-selective media used for the detection of V. dahliae in soil. Mycological Research, 107, 822–830. Hanlin, R.T. 1990. Illustrated genera of Ascomycetes. APS Press. Hoffmann, J.H., Moran, V.C., Impson, F.A.C. 1998. Promising results from the first biological control program against a solanaceous weed (Solanum elaeagnifolium). Agriculture, Ecosystems and Environment 70: 145-150. Ialongo, M.T. 1980. A population of Erysiphe trifolii on Securigera securidaca Degen et 3rd International Symposium on Weeds and Invasive Plants October 2-7, 2011 in Ascona, Switzerland Doerfler (fungi parasites of weeds). Annali dell’Instituto Sperimentate per la Patologia Vegetale, Rome. Mekki, M. Biology, distribution and impacts of silverleaf nightshade (Solanum elaeagnifolium Cav.) Bulletin OEPP/EPPO Bulletin 37, 114–118 Pickers, T. and Zimmermann, H.G. 1991. Biological control of Silverleaf nightshade, Solanum elaeagnifolium, and bugweed, Solanum mauritianum, (Solanaceae) in South Africa. Agriculture Ecosystems and Environment, 37: 137-155 Rector, B., Harizanova, V., Sforza, R., Widmer, T., Wiedenmann, R. N., 2006. Prospects for biological control of teasels, Dipsacus spp., a new target in the United States. Biological Control, 36, 1 - 14 Sheppard, A.W., Shaw, R.H., and Sforza, R. 2006. Top 20 environmental weeds for classical biological control in Europe: a review of opportunities, regulations and other barriers to adoption. European Weed Research Society Weed Research, 46: 93–117 Smith, C.G. 1969. Cross inoculation experiments with conidia and ascospores of Erysiphe polygoni on pea and other hosts. Transactions of the British Mycological Society, 53: 69-76. White, T. J., Bruns, T., Lee, S., and Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. (In M. A. Innis, D. H. Gelfaund, J. J. Sninsky, & White T. J. (Eds.), PCR Protocols: A guide to methods and applications 315322. San Diego: Academic Press).