Study on Sexual Fertility of Venturia inaequalis and Venturia pyrina under In vitro Conditions

Venturia species include many important plant pathogenic species such as Venturiainaequalis and V. pyrina causing scab or black spot on apple and pear. The Venturia teleomorph is the overwintering structure developing in leaf and fruit litters. The immersed teleomorphicpseudothecia are produced in the host tissues. This Ascomycetous genus is characterized by small, solitary, scattered or gregarious, globose, sub-globose, papillateascomata with setae around the ostiole. Asci are bitunicate, oblong to obclavate with a short pedicel or not. Ascospores ellipsoidal with broadly rounded ends, pale brown, one septate with two unequal size cells. Venturia has some species like V. inopina as homothallic and some like V. populina as heterothallic species. Sexual reproduction with producing sexual air-born spores plays an important role in distribution of disease in early spring. Therefore, awareness of sexual cycle in plant pathogenic fungi is very important due to management and forecasting probability of incidence of new genotypes (races). Venturia and related anamorphic species were collected from different places of Iran (18 provinces including Alborz, Ardabil, Eastern Azarbaijan, Golestan, Guilan, Hamadan, Isfahan, Kermanshah, Kurdistan, Lorestan, Markazi, Mazandaran, North Khorasan, Qazvin, Razavi Khorasan, Tehran, Western Azarbaijan, and Zanjan) during years 2013 and 2014. The collected specimens (800 specimens) were identified based onmorphological features. Isolation of the fungi from the infected samples was conducted using single spore method by streaking out spores on 2% water agar (WA) and culturing of single germinated conidium on potato dextrose agar (PDA). V. inaequalis was determined as a dominant species in Iran. Ten isolates from each of V. inaequalis and V. pyrina were crossed together under in vitro condition. Some isolates were cultured as single isolate in the petri dishes for investigation of homothallism and heterothallism. Several isolates from two species were crossed together to survey the biological species definition. Mature leaves of Golab apple cultivar (Malusdomestica) (for V. inaequalis isolates) and pear (for V. pyrina isolates) were washed under running tap water to remove dust and debris, and cut into 2×2 cm squares avoiding the midrib of the leaf. The leaf pieces were autoclaved and placed in petri dishes with 2% water agar. A plug of mycelium of each isolate was scraped from the edge of three weeks old colonies on PDA and mixed onto the leaf pieces with a scalpel. The plates were sealed with parafilm and placed in a sealed plastic bag. The cultures were incubated at room temperature under 12 hours light / 12 hours dark condition for 2 weeks, after which they were maintained, inverted in the dark at 8°C. The cultures were checked for the production of pseudothecia for the first time after six months. Among crossed isolates, seven and five fertile crosses were obtained from V. inaequalis and V. pyrina isolates, respectively, after six to eight months. In single isolate cultures, no sexual fertility was observed which confirm the heterothallic nature of both species. Moreover, between isolates from two species, no fertile crosswasobtained, which is the evidence of two distinct biological species. This is the first study on sexual reproduction of thesetwo species under in vitro condition in Iran. Morphological features of obtained sexual organs in vitro were the same as pseudothecia, asci and ascospores features in vivo. All ascospores in an ascus were fertile and they germinated on water agar after 24 hours. Based on this study, isolates with two different mating types are available as tester isolates. These isolates will be useful to design the primers to detect MAT alleles. PCR based methods can be used to study population genetic diversity, phylogeny, disease epidemiology and population biology (fungal species overwintering in a region based on mating types frequencies and their proportion). Development of reliable methods of sexual reproduction in vitroseems to be a valuable toolfor studies e.g. classic genetic analysis, strains modification and a supplement for modern genetic manipulations and evolutionary analysis. Our results verified heterothallism in both of V. inaequalis and V. pyrinaspecies. There was nofertile cross between isolates from two species that is the evidence of two distinct biological species. In this study, some tester isolates for mating typewere obtained which appear to be useful for the next PCR-based molecular studies on MAT alleles.