Diaporthe species infecting sunflower in Russia

Diaporthe is an important group of plant pathogenic fungi revealed all over the world. Early classification and species identification of this genus was mostly based on combination of morphological characteristics, cultural features, and affiliation with a host plant. According to recent investigations, valid distinction between Diaporthe species should have combined molecular techniques, morphological and cultural observations, and mating type data. In Russia a comprehensive and extensive analysis of biodiversity and geographic distribution of Diaporthe species infecting sunflower has not been performed. There were seven Diaporthe sp. strains isolated from this plant maintained in the Laboratory of Mycology and Phytopathology of All-Russian Institute of Plant Protection. In previous study a strain from Krasnodar region, based on combination of molecular and morphological features was identified as Diaporthe phaseolorum. The aim of this study was to identify all other strains using primarily molecular phylogenetic approach and traditional morphological analysis. The strains were identified as Diaporthe gulyae, Diaporthe eres, and Diaporthe helianthi. Two species D. gulyae and D. eres are found for the first time on sunflower in Russia. Detection of D. helianthi is the first report of this fungus in Russia as confirmed by molecular analysis.


Introduction
Fungal species belonging to Diaporthe Nitschke are the most common and widespread fungi worldwide. This genus is extremely large and species are known as saprotrophs, endophytes, predominately phytopathogens, causing leaf and stem spots in a broad range of economically important agricultural crops, including sunflower [2]. Phomopsis stem canker is ubiquitous in many sunflower-producing regions [3,4,5]. This disease is one of the primary limiting factors for sunflower production in Europe, where yield losses reached up to 50% and losses in oil content exceeded 10% [4]. The pathogen causing Phomopsis stem canker on Helianthus annuus was initially described as Diaporthe helianthi Munt.-Cvetk., Mihaljc. & M. Petrov (anamorph synonym -Phomopsis helianthi Munt.-Cvetk., Mihaljc. & M. Petrov) in the former Yugoslavia in 1980. All Diaporthe spp. isolated from infected sunflower were assumed to be D. helianthi. Although D. helianthi was described as the common causal agent, the possibility of multiple species infecting sunflower was raised in the early 1980s [6]. However, there was little evidence to support this hypothesis. At that time identification of Diaporthe spp. was based on morphological and cultural features and on association with a certain host [7]. Since the description of D. helianthi, recent investigations [5,[8][9][10][11][12], employing multi-locus molecular phylogenetic approach analyses of DNA sequence data of the ITS, β-tubulin (TUB), translation elongation factor-1α (TEF) genes resulted in resolving additional at least 12 Diaporthe species, which together with including D. helianthi could be harmfull for sunflower. The study of the biodiversity and geography of the Diaporthe species associated with H. annuus in Russia according to actual taxonomy of the genus with introduction of molecular techniques just getting started. To the moment there is a single record, which has been verified by molecular phylogenetic features. This is report about Diaporthe phaseolorum (Cooke & Ellis) Sacc. on sunflower in Krasnodar region [1]. The objective of this study was to correctly reidentify isolates of Diaporthe sp. from seeds and stems of sunflower originated from the Saint Petersburg, Belgorod and Stavropol regions.

Isolates
Survey of 50 sunflower fields in 10 regions of Russia (Altay, Krasnodar, Stavropol territories, Belgorod, Lipetsk, Penza, Rostov, Tchelyabinsk, Tula regions and Saint Petersburg) has been conducted. Twenty-two samples of seeds derived from nine regions of Russia (Astrakhan, Orlyol, Belgorod, Tchelyabinsk, Tambov, and Volgograd regions as well as Altay, Krasnodar and Stavropol territories) has been analyzed. Six Diaporthe strains were collected from stems and seeds (table 1). To isolate a pure culture of fungus from the sunflower, fragments of material were surface sterilized with 20 ml of 2% sodium hypochlorite (NaClO) solution. After the surface sterilization, the samples were placed on potato sucrose agar (PSA) [13] containing antibiotics (100 μg/ml ampicillin, streptomycin, penicillin, HyClone™, GE Healthcare Life Science, Austria) and 0.4 μl/l Triton X-100 (Panreac, Spain) that restricts the growth of fungi. The Petri dishes were incubated at 24°C in the dark and were analyzed on the 7-10th day of cultivation. Samples of infected stems were deposited in the Mycological Herbarium (LEP) of All-Russian Institute of Plant Protection (VIZR). All Diaporthe isolates were stored in plastic microtubes on PSA at + 4°C in the VIZR pure culture collection.

Results
In all phylogenetic trees inferred from ITS, TUB, and TEF, as well in combined trees, six studied Diaporthe sp. isolates clustered in order Diaporthales, family Diaporthaceae with the highest value of bootstrap support (Fig. 1). Two strains (MF-Ha18-001, MF-Ha18-002) in a combined tree, inferred from all three loci, formed one clade with reference strains of D. eres Nitschke species group, but they were outside the subclade, which contain the type culture of D. eres. Whereas in the phylogram, based on TUB and TEF genes these two strains joined together in one clade, closely related to that with type D.

Discussion
Currently it is widely known, that species identification in Diaporthe should be implemented using a polyphasic approach (Consolidated Species Concepts (CSC)) and based on phylogenetic, morphological, and biological characteristics. A multi-locus phylogenetical analysis should be based on combined DNA data matrix of ITS and partial sequences from TUB and TEF genes. Six Russian Diaporthe strains isolated from sunflower based on molecular phylogenetic data were identified as D. gulyae, D. eres and D. helianthi. Diaporthe gulyae was found on sunflower seed originated from Belgorod region. Diaporthe eres was detected on sunflower stems from Saint Petersburg. And finally D. helianthi, that was previously treated as the only Diaporthe species ordinary infected sunflower in Russia, was found only in Stavropol territory. Findings of two species, D. gulyae and D. eres, are the first reports of these fungi on sunflower in Russia. Detection of D. helianthi confirmed by molecular phylogenetic analysis is the first valid identification of this species in the country. Previous reports of D.
helianthi have been based only on morphological features or symptoms on sunflower stems and leaves.