Aeciospores of Gymnosporangium confusum formed in aecia on the lower leaf surface of Crataegus monogyna and in stromatic structures in overwintered galls showing surface ornamentation 

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... ğ lu and Yildiz 2005) and in the Mediterranean parts of Europe in the 1950 ’ s (Bernaux 1956). Hawthorn ( Crateagus spp.) is a native plant of the Mediterranean regions of North Africa, Europe, and central Asia, and now grows in many areas of North America. Turkey is one of the centers for genetic diversity of Crataegus (Ercisli 2004). Recently, hawthorns have been cultivated as a horticultural crop for their fruits and also for medicinal purposes in several parts of Turkey and distributed to other provinces. It is reported that hawthorn fruits have high flavonoid, vitamin C, glycoside, anthocyanin, saponin, tannin, and antioxidant levels (Ljubuncic et al. 2005) as well as phenolics (Chang and Zuo 2002; Schussler and Holzl 1995). The objectives of this study were to (i) identify hawthorn rusts in the orchards and forest areas in Hatay province, Turkey, based on morphology and analyses of DNA sequence data; and (ii) document the rust life cycles for disease control. Morphological identification Fruit orchards and forests in Alt ı nözü, Antakya, Belen, Samanda ğ , and Yaylada ğı districts of Hatay province were surveyed from 2007 to 2009. Leaf lesions and branches with galls from Crataegus monogyna , C . orientalis, Cydonia oblonga, Malus domestica L., Pyrus communis L. (Rosaceae), and Juniperus spp. (Cupressaceae) were collected and brought to the laboratory in paper bags between February and August each year. In 2007 and 2008, only galls were sampled. Tissues of infected parts were boiled in 10% KOH solution for about 5 min and slides were prepared for morphological identification of the causal agent. Then 30, 20, 20 and 25 trees of C. monogynya, C. orientalis, P. communis and Juniperus spp., respectively, were selected from rust infection-positive areas in all locations. The specimens from those trees were coded and labeled based on the sampling district and tree. After identification, records from field and laboratory observations were gathered for the respective species. Therefore, the occurrence of each species was determined with the life stage of the pathogen on infected plant parts on each date surveyed; these data are presented in Table 1. Molecular identification Tissue with rust symptoms from C. monogyna , C. orientalis , J. communis, and P. communis were sent to the USDA Systematic Mycology & Microbiology Laboratory in Beltsville, MD, for molecular identification. A small amount of symptomatic tissue from each sample was added to bead solution tubes from the Mo Bio UltraClean Plant DNA extraction kit (Carlsbad, CA, USA). Samples were processed twice in a FastPrep (Bio101, Vista, CA, USA) for 30 s at 5.0 m s − 1 with 10-min room- temperature incubations between runs. Genomic DNA was extracted according to the manufacturer ’ s spec- ifications except that tubes were incubated at 55°C overnight in P1 solution. Primer pairs Rust2inv (Aime 2006) and LR6 (Vilgalys and Hester 1990) were used to PCR (polymerase chain reaction) portions of the ITS and nLSU gene regions and products were sequenced with an ABI 3130 (Applied Biosystems, Foster City, CA, USA) using the PCR primers and LR3 (Vilgalys and Hester 1990) and LROR (Moncalvo et al. 1995). Sequences were assembled and edited using Sequencher 4.8 (Gene Codes, Madison, WI, USA) and compared with those from Gymnosporangium species in GenBank. Based on morphological and microscopic analysis of rDNA sequence data, three species were identified, namely, G . clavariiforme on C . orientalis, G . confusum on C . monogyna , and G . sabinae on P . communis . G. confusum was also found on J. communis . Other members of the Rosaceae, including M . domestica and C . oblonga, were not found to be infected by species of Gymnosporangium . Sequences from the ITS region and the 28S ribosomal RNA gene were deposited in GenBank: G. confusum (GU058011, HM114219), G. clavariiforme (HM114220), G. sabinae (HM114221). DNA sequence data of the Gymnosporangium rust collected on C. monogyna matched the Gymnosporangium rust from Juniperus spp. This rust was identified as G. confusum based only on morphology, since there were no G. confusum sequences on GenBank, prior to this collection. Morphological features of the Gymnosporangium rust from C. orientalis were consistent with G. clavariiforme and two point mutations separate this isolate from t h e G . c l a v a r i i f o r m e i s o l a t e in G e nB a n k (AF426211). Morphological features of the Gymnosporangium rust from P. communis were consistent with G. sabinae and two point mutations separate this isolate from the two G. sabinae sequences in GenBank (AY512845, AF426209). sporangium spp . Gymnosporangium confusum In February, some gall formations were observed on C . monogyna (wild hawthorn) (Fig. 1). In the first half of March (Table 1), leaf and flower spots caused by G. confusum were observed on the same host. Upon microscopic examination, aecia containing aeciospores were found on lower leaf surfaces and in galls (Fig. 2). Primary infections were caused by basidiospores released from galls on J. communis (Fig. 3) in the first week of April. Infections occurred mainly on newly developed leaves, flowers and fruits (Fig. 4) of C. monogyna . Symptoms developed on the upper sides of leaves; then spermogonia with few spermatia developed in the center of these lesions (Fig. 5). Aecia developed on the underside of these lesions. In April, aecial peridia developed and aeciospores were released to potentially infect J. communis (Fig. 6). Aecial horns lacking a peridium began to develop in the beginning of May and were fully formed and releasing aeciospores in approximately 10 days (Fig. 7). Galls containing aecia on hawthorns apparently overwintered (Figs. 8 and 9). In May June, new galls were detected on J. communis containing few teliospores but lacking horn development. Junipers with small, thin galls were found on nearby wild and cultivated hawthorns and apple trees. Galls containing telia had tongue-shaped gall cracks (Fig. 3), although teliospores were sparse (Fig. 10). Teliospores were two-celled, ellipsoid, brownish orange, and 38 – 46×25 – 34 μm. Ribosomal DNA sequences from these galls matched those isolated from C. monogyna . Gymnosporangium clavariiforme Infections caused by G. clavariiforme were not observed until the second half of April on C. orientalis . Primary leaf infections were caused by basidiospores released from neighboring junipers. Infections occurred mainly on newly developed leaves and flowers of C . orientalis . Symptoms were observed on both upper and lower leaf surfaces, although aecia were more prominent on lower leaf surfaces (Fig. 11). In May, gall formation occurred on C. orientalis from infections initiated in April. In early June, horns began developing on these galls, and aecia were released in approximately 10 – 15 days, potentially causing infections on J. communis (Fig. 12). Gymnosporangium sabinae In all surveyed areas, the first disease symptoms on P. communis were caused by the spermogonial stage of G. sabinae and were observed in late April. The aecial stage did not form until October. Infections of P. communis by G. sabinae were observed about 2 months later than those observed on hawthorn, caused by G. communis and G. clavariiforme . Sequence data from the nuclear ribosomal ITS2 and the 5 ′ portion of the LSU locus were useful as barcodes for identifying G. clavariiforme and G. communis to species. Identification of G. confusum was based on sequence data (M. C. Aime, personal communication) and morphology since there were no G. confusum sequences deposited to GenBank prior to this study. Gymnosporangium clavariiforme and G. confusum produced overwintered aecial galls on their respective secondary hosts, namely, C. orientalis and C. monogyna . Aecia were produced within these overwintered galls from April to June. Additional infections were observed in May on fruits and leaves of Crataegus , presumably caused by the additional release of basidiospores from Juniperus spp. The absence of teliospore horns on junipers in this study differs from previous reports describing numer- ous teliospore horns on ornamental juniper plants and major rust epidemics in Turkey and Europe (Erdo ğ du et al. 2010; Kern 1973; Peterson 1982). Only few teliospores were found inside gall cracks on juniper branches, although one would expect to find telial horns abundantly in May – October. Although G . confusum and G . clavariiforme have been reported to occur on apple and quince (Dinç and Yilmaz 1978), we did not find rust symptoms on these species. The known host range of G. confusum includes Cotoneaster spp., Crataegus spp., Cydonia spp., Eriobotrya japonica (Thunb.) Lindl., Juniperus spp., Malus spp., Pyracantha coccinea M. Roem., Pyrus spp., and Sorbus spp. The known host range of G. clavariiforme includes Amelanchier spp., Aronia arbutifolia (L.) Pers., Cotoneaster spp., × Crataego- mespilus spp. ( Crataegus × Mespilus ), Crataegus spp., Cydonia spp., Juniperus spp., Malus sylvestris (L.) Mill., Pyrus spp., and Sorbus spp. (Farr and Rossman 2010). These hosts include widely distributed ornamentals and indigenous plants in areas surround- ing the cultivation of rosaceous plants. Since their host ranges overlap, diagnostic features distinguishing the two common species of Gymnosporangium of Crataegus in Turkey are important for accurate identification. An understanding of the life cycles of these fungi may help control diseases caused by Gymnosporangium on economically important Rosaceae. In 2007 and 2008 one species of megastigmine wasp, Westalianus altinozus Do ğ anlar (Hymenoptera, Torymidae), one Sinoxylon sp. (Coleoptera, Bostry- chidae), and some unidentified lepidopterous larvae were found feeding inside the galls on C. monogyna . Westalianus altinozus was found as a new parasitic species (Do ğ anlar 2010) on the caterpillars of Microlepidoptera. Sinoxylon spp. move between trees for ...