Samina Sarwar

Muhammad Hanif
email: mhanif_r@hotmail.com

A N Khalid

Jacques Guinberteau
email: guinbert@bordeaux.inra.fr

DIVERSITY OF BOLETES IN PAKISTAN -FOCUS ON SUILLUS BREVIPES AND SUILLUS SIBIRICUS

Keywords: Ectomycorrhizae, Non-gilled fungi, Pakistan, PCR-ITS, Sequence alignment

During the exploration of diversity of non-gilled fungi and their ectomycorrhizal morphotypes from Pakistan, Suillus brevipes was found ectomycorrhizal with Quercus incana while Suillus sibiricus was found associated with roots of Pinus wallichiana and Salix alba. Basidiomes and ectomycorrhizae of the latter are characterized morpho-anatomically and by using rDNA-ITS and 5.8S gene while for the former, both the basidiome and the ectomycorrhizae have been identified and characterized morpho-anatomically. Previously, S. sibiricus was found associated only with P. wallichiana. In the present work S. alba has been found as new host for S. sibiricus.

Molecular analysis of S. sibiricus was used to describe the phylogenetic position of the fungus using Maximum Parsimony analysis. S. brevipes and morphotypes of both boletes are new records for Pakistan.

INTRODUCTION

Boletes (Basidiomycota; Agaricomycetes) are characterized by the presence of tubes instead of gills [START_REF] Thiers | California Mushrooms. A field guide to the boletes[END_REF][START_REF] Wood | The Boletes of California[END_REF]. There are 35 genera and more than 1,000 species of Boletes known worldwide [START_REF] Kirk | Dictionary of the Fungi[END_REF] with only ten [START_REF] Halling | Pacific boletes: implications for biogeographic relationships[END_REF] genera and forty four (44) species reported from Pakistan [START_REF] Ahmad | Fungi of Pakistan[END_REF][START_REF] Razaq | Taxonomic Studies on Basidiomycota from Northern Areas of Pakistan[END_REF][START_REF] Niazi | Biodiversity of Ectomycorrhizas in Conifers from Himalayan Moist Temperate Forests of Pakistan[END_REF]. They are distributed mostly in Europe, Asia, North America, in the temperate latitude in the northern hemisphere [START_REF] Corner | Boletus in Malaysia[END_REF][START_REF] Wu | Biogeographic relationships between the macrofungi of temperate eastern Asia and eastern North America[END_REF] while mostly confined to moist temperate coniferous forests in Pakistan [START_REF] Ahmad | Fungi of Pakistan[END_REF][START_REF] Ahmad | A contribution to the Agaricales of Pakistan[END_REF]. More than 90% of members of this group form symbiotic associations with the root system of forest trees, which is the most important tool for germination and good development throughout the life of tree [START_REF] Halling | Pacific boletes: implications for biogeographic relationships[END_REF]. The plant families Betulaceae, Caesalpiniaceae, Casuarinaceae, Dipterocarpaceae, Ericaceae, Fagaceae, Mimosaceae, Myrtaceae, Pinaceae and Salicaceae form ectomycorrhizal symbioses with Boletes [START_REF] Newman | The distribution of mycorrhizas among families of vascular plants[END_REF][START_REF] Lee | Ectomycorrhizas and putative ectomycorrhizal fungi of Shorealeprosula Miq. (Dipterocarpaceae)[END_REF]. This group is economically important because it contains edible, medicinally-important and poisonous mushrooms [START_REF] Benjamin | Red-pored boletes. In: Mushrooms: poisons and panaceas -a handbook for naturalists, mycologists and physicians[END_REF][START_REF] Hall | Ectomycorrhizal fungi with edible fruiting bodies. 2. Boletus edulis[END_REF][START_REF] Lovy | Activity of edible mushrooms against the growth of human T4 leukemic cancer cells, HeLa cervical cancer cells, and Plasmodium falciparum[END_REF]. Suillus P. Micheli ex Adans. is a genus of Boletes belonging to Suillaceae represented by 54 species worldwide [START_REF] Kirk | Dictionary of the Fungi[END_REF] with only four species known from Pakistan [START_REF] Ahmad | Fungi of Pakistan[END_REF][START_REF] Razaq | Taxonomic Studies on Basidiomycota from Northern Areas of Pakistan[END_REF][START_REF] Niazi | Biodiversity of Ectomycorrhizas in Conifers from Himalayan Moist Temperate Forests of Pakistan[END_REF]. Wu et al. [START_REF] Wu | Phylogenetic and biogeographical relationships of eastern Asia and eastern North American disjunct Suillus species (fungi) as inferred from nuclear ribosomal RNA ITS sequences[END_REF] discussed the bio-geographic pattern and phylogenetic relationship of Suillus species from Eastern Asian (China and Nepal) and North American territories. During an investigation of the Section: Diversity and Taxonomy 124 diversity of boletes in Pakistan, S. brevipes and S. sibiricus were isolated and in the present work they are described morpho-anatomically along with their ectomycorrhizal morphotypes from Pakistan. Suillus sibiricus and its ectomycorrhizae have been described up to molecular level and identified by using nrRNA tandem repeats. Molecular phylogeny of S. sibiricus has also been discussed with Eastern Asian and Eastern North American isolates retrieved from Genbank.

MATERIALS AND METHODS

The study area is located in Himalayan moist temperate forests of Pakistan that are evergreen forests of conifers mixed with some oaks and other deciduous broad-leaved trees. Their undergrowth is rarely dense, and consists of both evergreen and deciduous species. These forests occur between 1,500 m and 3,000 m elevation with mean annual rainfall about 1,700 mm. Average aerial humidity is 57% and the mean annual temperature is 12ºC [START_REF] Siddiqui | COUNTRY REPORT -PAKISTAN[END_REF]. Many Boletes form symbiotic association with the roots of these plants for their better growth [START_REF] Ahmad | Fungi of Pakistan[END_REF].

Sampling was done during the rainy season from July to August 2008 -2010. Sporocarps and soil samples containing their ectomycorrhizal morphotypes were collected. Small parts of both the fruiting body and ECM were stored in 2% CTAB buffer for DNA analysis. Soil blocks were wrapped in polythene bags to avoid evaporation and crumbling. Fresh characters of sporocarps were recorded in the field and photographed. Sporocarps were dried properly and the soil samples were washed to separate the ECM. Both sporocarps and ECM were characterized morpho-anatomically. The identification was done with the help of available literature [START_REF] Mcknight | A field guide to Mushrooms: North America[END_REF][START_REF] Santana | Boletes from Belize and Dominican Republic[END_REF][START_REF] Scates | Trial field key to the Boletes in the Pacific Northwest[END_REF]. The nomenclature was made according to the Index Fungorum. Special designation numbers were given to each of the samples. The specimens were deposited in Punjab University herbarium, Botany department (LAH). Molecular Analysis. DNA was extracted by modified CTAB method following Gardes & Bruns [START_REF] Gardes | Methods in Molecular Biology. Species Diagnostics Protocols: PCR and Other Nucleic Acid Methods[END_REF]. The hymenial tissue was removed with sterile forceps and rinsed with sterile H 2 O. Fresh and healthy ectomycorrhizal morphotypes from Pinus wallichiana A.B. Jack. and Salix alba L. were selected manually under a stereomicroscope (Olympus, SZ30, Japan). The extraction was modified for silica emulsion binding and purification (Gene-Clean; Q-Biogene, Irvine, CA, USA). The ITS regions of nuclear rDNA were amplified by Polymerase Chain Reaction (PCR) using universal primers i.e. ITS1 and ITS4 primers [START_REF] Gardes | Methods in Molecular Biology. Species Diagnostics Protocols: PCR and Other Nucleic Acid Methods[END_REF][START_REF] White | Amplification and Direct sequencing of Fungal ribosomal RNA genes for Phylogenetics. PCR Protocol, a Guide to Methods and Applications[END_REF]. Purified PCR products were sequenced using a DNA sequencer (3730ABi). DNA sequence was submitted to BLAST and used to query the nucleotide collection using default settings. rDNA-ITS sequences of S. sibiricus were submitted to GenBank. Phylogenetic position of S. sibiricus was deduced by Maximum Parsimony with bootstrapping using PAUP* Version 4.0b10. For that purpose, a tree was made by using seven [START_REF] Corner | Boletus in Malaysia[END_REF] sequences obtained from S. sibiricus from Pakistan and 23 closely related rDNA ITS sequences of Suillus spp. were retrieved from the DNA database using the BLAST program from GenBank (Table 1). All sequences were aligned and corrected manually using MacClade 4.08 and Bioedit (version 7.0.9).

Section: Diversity and Taxonomy 125

RESULTS

Suillus brevipes (Peck) Kuntze, Revis. gen. pl. (Leipzig) 3(2): 535 (1898) (Fig. 1. A-F). Pileus convex to hemispheric, 2.5-6 cm, chocolate brown, shiny, smooth, glabrous, sticky, flesh thick and off-white, margins slightly incurved, entire, smooth, of same color like pileus surface. Hymenium adnate and ascending, frequent pores, creamish to light yellowish, no color change upon bruising, pores rounded to irregular in shape. Stipe centric, clavate, 3.5-6.8 cm long, 1.3-2.2 cm wide, smooth, whitish with brown small patches at some points, semi-hollow, ring or volva absent. Basidia clavate, thin walled, contents brown in Meltzer's, 18-26 x 6-8 µm, 2-4 sterigmate. Basidioles clavate, thin walled, 15-23 x 5.5-8 µm. Cystidia cylindrical to clavate to subfusoid to ampullaceous, thick walled, dark brown contents, 35-49 x 5-9 µm. Basidiospores ellipsoid to fusiform to subfusiform, smooth, thick walled, 6-10 x 3-6.5 µm, honey brown to light yellowish.

Description of ectomycorrhizae of Suillus brevipes

Morphological characteristics: (Fig. 2. A & B). Mycorrhizal system: monopodial pinnate, system 3.5-4 mm long, main axis 0.5 mm wide, texture smooth, tips beaded to straight, tip length about 0.5 mm and <0.5 mm wide, young tips honey brown, older tips black and apices light honey brown, host tissue visible under mantle surface. Rare Rhizomorphs, at restricted points, whitish brown. Common emanating hyphae, straight, whitish. Anatomical characteristics of mantle in plan views (Fig. 2. D & E). Plectenchymatous outer mantle layer (type E, Agerer) [START_REF] Agerer | Colour Atlas of Ectomycorrhizae (1st-11th del)[END_REF], matrix material granular, cells 3 µm wide and 11 µm long, honey brown, cell contents: granular, common septa and clamped septa, hyphal junction angle about 60˚, rare anastomose. Plectenchymatous inner mantle layer (type E, Agerer) [START_REF] Agerer | Colour Atlas of Ectomycorrhizae (1st-11th del)[END_REF], granular cell contents, cells 5 µm wide and 12 µm long, yellowish brown, common septa and clamped septa, common hyphal junction, hyphal junction angle about 45˚, rare anastomose and H-shaped. Anatomical characteristics of emanating elements (Fig. 2. C & F). Rhizomorphs highly differentiated (type B, Agerer) [START_REF] Agerer | Colour Atlas of Ectomycorrhizae (1st-11th del)[END_REF], cells 4 µm wide and 9 µm long, light brown in color, cell contents oil like bodies, common septa and clamped septa, common hyphal junction and Yshaped, anastomose H-shaped. Rare Emanating hyphae, cells 5 µm wide and 8 µm long, reddish brown, septa and clamped septa present.

Suillus sibiricus (Singer) Singer, Farlowia 2: 260 (1945) (Fig. 3. A-F) MYCOPAK: EA15194, EA23904, EA24040, EA24104, EA17193, SS142010, SS022010. GENBANK: JN119748-54 Pileus 4-8 cm, pulvinate to obtuse, sticky, slimy, shiny, glabrous, yellowish brown, smooth, margins smooth, entire, slightly darker color than pileus surface, deflexed. Hymenium: adnate and ascending, pores frequent, yellowish, angular pores. Stipe: 10 cm long, 1.8 cm wide, central, equal, ring present, yellowish to off-white from apex to ring, reddish brown from ring towards base, whitish near base, reticulated, reticulations white, rough, solid, curved. Basidia: long clavate, 2-4 sterigmate, thick walled, brownish contents visible in Meltzer's, 25-39 x 7-9 µm. Basidioles: clavate, thick walled, contents visible, (20-) 26-31 x 7.5-8 µm. Cystidia: cylindrical to subfusiform, thick walled, yellowish brown in Meltzer's, yellowish brown contents, 33-45 (-62) x 7-9 µm. Pileocystidia: a tangled layer of repent hyphae, hyaline to light honey in Meltzer's, thin walled, granular contents, septate, 60-74 x 8-10 µm. Caulocystidia: long, subclavate-clavate-cylindrical, some with pointed ends, septate, thick walled, hyaline in Meltzer's, 68-99 x 13-20 µm. Basidiospores: ellipsoid-fusiform, thin walled, amyloid, smooth, 10-12 x 3.5-5 µm.

Description of Ectomycorrhizae of Suillus sibiricus with Pinus wallichiana

Morphological Characters (Fig. 4. A & B). Ectomycorrhizal system frequently found under the fruiting bodies, dichotomously branched, 3-5 mm long, 0.5 mm in diam., length of tips 1.5 mm, dark honey brown when older. Very tips honey brown to reddish brown, tips straight. Mantle surface long-spiny to cottony; host tissue not visible under the sheath. Rhizomorphs: Section: Diversity and Taxonomy 127 common, connecting distinctly to the mantle surface, off-white to light brown, differentiated. Emanating hyphae: light brown, common, giving cottony appearance, straight. Anatomical characteristics of mantle in plan views. (Fig. 4. C & D). Mantle: pseudoparenchymatous in all layers. Outer mantle layer: pseudoparenchymatous (type L, Agerer) [START_REF] Agerer | Colour Atlas of Ectomycorrhizae (1st-11th del)[END_REF], cells rounded to angular, cells 7-9 µm long, 7.3-10 µm in diam., no matrix material observed, cell contents clear, cell color hyaline to light yellowish. Inner mantle layer: pseudoparenchymatous (type H; Agerer) [START_REF] Agerer | Colour Atlas of Ectomycorrhizae (1st-11th del)[END_REF], without any ring like structures; cells colorless to yellowish, cells 6.5-7.4 µm in diam., cell walls 5.4-7.2 µm thick; cell contents not observed. Morphological characteristics (Fig. 5. A). Mycorrhizal system found under the fruiting bodies: monopodial pinnate, system up to 5.5 mm long, with 0.6 mm thick main axis, unramified tips 2.2 mm long and about 0.5 mm thick, color of system dark brown, younger tips honey brown, rounded and slightly curved, surface of mycorrhizal system smooth, host tissue visible under mantle surface. Rhizomorphs: rare, attached at restricted points, dark brown to black. Molecular identification and characterization of S. sibiricus and its ectomycorrhizae. PCR-ITS products of rDNA obtained from basidiomes and their ectomycorrhizae with Pinus wallichiana and Salix alba were sequenced. The sequences showed 99% similarity with isolates of S. sibiricus from China and America, confirming the morphological identification. The phylogenetic analysis for genus Suillus was carried out using parsimony as optimality criterion. The sequences included in this analysis had around 659 characters, from which 494 characters were used for further analysis after alignment and trimming from both 3' and 5' sites of rDNA-ITS. After that, none of characters were excluded from final analysis.

All characters were of type 'unord'. There were 53 parsimony-informative sites, 415 constant sites, 26 variable characters were parsimony-uninformative. All the gaps were treated as "missing" data. Multistate taxa were interpreted as uncertainty. The starting tree(s) was obtained via stepwise addition with random addition of sequence and 1000 number of replicates. There were 49145891 starting seeds for the tree generated. Only 01 tree held at each step during stepwise addition of the sequences. Tree-bisection-reconnection (TBR) was used as branchswapping algorithm. A total of 6,457,051 rearrangements were tried for the best tree. Only 27 trees were retained for analysis. The genetic distance matrix was derived from Maximum Parsimony (MP) analysis generated a consensus tree from the best 144 trees showing the following scores: Tree length (TL) = 146, consistency index (CI) = 0.6438, homoplasy index (HI) = 0.3562, CI excluding uninformative characters = 0.5517, HI excluding uninformative characters = 0.4483, retention index (RI) = 0.7977, rescaled consistency index (RC) = 0.5136. Phylogenetic analysis showed the various species of Suillus. Maximum Parsimony consensus tree indicated three major clades and one independent clade. Maximum Parsimony consensus tree was constructed exclusively for Suillus species from geographically different localities, mostly from Eastern Asia (China and Nepal), Eastern North America and from Pakistan to resolve exact identification. The cladogram represents a major polytomous clade formed by S. americanus and S. sibiricus species (Table 01). All of the species of this clade shared 98-99% of characters studied so far for this analysis and thus identified as S. sibiricus. Both S. sibiricus and S. americanus occupied topologically different positions in the same polytomous clade (Fig. 6).

The Maximum Parsimony analysis resulted in major polytomous clade comprising sixteen isolates of S. americanus and S. sibiricus. All these species are monophyletic along with S. flavidus (Accession No. FJ845439.1), S. megaporinus (Accession No. GQ249400.1) and S. umbonatus (Accession No. L541115.1).

DISCUSSION

Boletes are an important component of Himalayan Moist Temperate Forests of Pakistan and make ectomycorrhizal association with gymnosperm and angiosperm trees of this area. Out of ten [START_REF] Halling | Pacific boletes: implications for biogeographic relationships[END_REF] genera of 44 species of Boletes, Suillus is represented by 4 species viz. S. granulatus (L.) Roussel, S. placidus (Bonord.) Singer, S. sibiricus (Singer) Singer and S. tomentosus (Kauffman) Singer [START_REF] Ahmad | Fungi of Pakistan[END_REF][START_REF] Razaq | Taxonomic Studies on Basidiomycota from Northern Areas of Pakistan[END_REF][START_REF] Niazi | Biodiversity of Ectomycorrhizas in Conifers from Himalayan Moist Temperate Forests of Pakistan[END_REF].

Suillus is very important genus of Boletes. Its phylogenetic and bio-geographic relationship comprehensively described by Wu et al. [START_REF] Wu | Phylogenetic and biogeographical relationships of eastern Asia and eastern North American disjunct Suillus species (fungi) as inferred from nuclear ribosomal RNA ITS sequences[END_REF]. Suillus sibiricus was originally described by Rolf Singer in 1938 [START_REF] Kuo | Suillus sibiricus. Retrieved from the MushroomExpert[END_REF]. While S. brevipes was first described by Charles Frost in 1874 [START_REF] Halling | Boletes Described by Charles C. Frost[END_REF].

In the present studies S. brevipes and S. sibiricus have been found forming ectotrophic mycorrhizae with different host trees of Himalayan Moist Temperate Forests of Pakistan. The Section: Diversity and Taxonomy 131 S. brevipes has similarity with S. granulatus, S. albidipes and S. pallidiceps. But the major differences from these species are S. granulatus has shorter stem, and distinct raised granules on the stem while S. brevipes has smooth white stipe. Similarly, S. brevipes is differentiated from S. albidipes by not having a cottony roll of velar tissue at the margin when young. A major difference of S. brevipes from S. pallidiceps is a pale yellow cap color of the latter while the former has a chocolate brown cap [START_REF] Tylukti | Mushrooms of Idaho and the Pacific Northwest[END_REF]. Previously, it was found associated with Pinus contorta and P. ponderosa [START_REF] Grand | Conifer associated and mycorrhizal syntheses of some Pacific Northwest Suillus species[END_REF] and there is no report regarding their distribution in South Asia. The mycorrhizae of S. brevipes were described with P. contorta which form tuberculate system and tortuous tips. While in present study, with Q. incana it forms monopodial pinnate and possess plectenchymatous type of mantle which is in contrast with synchymatous type of mantle in P. contorta.

S. sibiricus often confused with North American species, S. americanus. The latter has larger sporocarps as compared with S. sibiricus. Despite this minor difference, other macro and micro features resemble each other. The only thing separating them is their geographical distribution [START_REF] Wu | Phylogenetic and biogeographical relationships of eastern Asia and eastern North American disjunct Suillus species (fungi) as inferred from nuclear ribosomal RNA ITS sequences[END_REF]. Suillus flavidus and S. umbonatus are also close relatives of S. sibiricus that form sister clade with S. sibiricus in constructed Cladogram (Fig. 6). The ectomycorrhizae of S. sibiricus were previously described with Pinus cembra, P. banksiana, P. monticola, P. peuce, P. pumila and P. sibirica [START_REF] Kuo | Suillus sibiricus. Retrieved from the MushroomExpert[END_REF][START_REF]Maslak syberyjski Suillus sibiricus[END_REF][START_REF] Grand | Conifer associates and mycorrhizal synthesis of some Pacific Northwest Suillus species[END_REF][START_REF]Maslyonok Sibirskiy (Suillus sibiricus)[END_REF][START_REF] Snell | Notes on Boletes. XIV[END_REF][START_REF] Dahlberg | 33 Threatened Fungi in Europe[END_REF]. According to Agerer [START_REF] Agerer | Colour Atlas of Ectomycorrhizae (1st-11th del)[END_REF], S. sibiricus forms coralloid mycorrhizal with P. cembra. Mantle is typically of plectenchymatous type with club-shaped cells. Hyphae of mantel embedded in the pigment matrix and possess large hyphae in During present investigation, it is concluded that S. brevipes and its ectomycorrhizae are new reports for Pakistan. In addition, molecular analysis of S. sibiricus and its ectomycorrhizae are being done for the first time in Pakistan and association of this fungus with Salix alba is new to science world and it seems that S. sibiricus extends its host range from conifers to deciduous trees.

Figure 1 .

 1 Figure 1. Suillus brevipes, A-F. Views of the Sporocarp (A & B), Basidia (C), Basidioles (D), Cystidia (E), Basidiospores (F). Scale Bars for A & B = 1 cm, C = 6 µm, D = 6 µm, E = 8 µm, F = 3 µm.

Figure 2 .

 2 Figure 2. A-F. Ectomycorrhizae of Suillus brevipes. Views of ECM under stereomicroscope (A & B). Anatomical features of ECM under light microscope (C-F). Rhizomorph (C), Outer mantle (D), Inner mantle (E), Emanating hyphae (F). Scale Bars for A & B = 0.6 mm, C = 4.5 µm, D = 7 µm, E = 7.5 µm, F = 5 µm.

  Anatomical characteristics of emanating elements (Fig. 4. E & F) Rhizomorphs: highly differentiated, cells about 5.4 µm in diameter and about 87 µm length, light brown color, septa rare, clamps absent, cell contents clear. Emanating hyphae: common, 5 µm in diameter, 67 µm long hyphal cells, only clamped septa present. Habitat and Distribution of Suillus sibiricus. On ground, in groups, under Pinus wallichiana, at 2347 m. a. s. l., Pakistan, KPK, Khera gali, 7 th August, 2010. S. S. B. # 53.

Figure 3 .

 3 Figure 3. Suillus sibiricus, A-F. Views of the Sporocarp (A & B), Basidia (C), Basidioles (D), Cystidia (E), Basidiospores (F), Pileocystidia (G), Caulocystidia(H) . Scale Bars for A & B = 1.4 cm, C = 7 µm, D = 7 µm, E = 8.5 µm, F = 4.5 µm, G = 12 µm, H = 24 µm.

Figure 4 .

 4 Figure 4. A-F. Ectomycorrhizae of Suillus sibiricus with Pinus wallichiana. Views of ECM under steriomicroscope (A & B). Anatomical features of ECM under light microscope (C-F). Outer mantle (C), Inner mantle (D), Rhizomorph (E), Emanating hyphae (F). Scale Bars for A & B = 1 mm, C = 7.5 µm, D = 10 µm, E = 16 µm, F = 9.5 µm.

  Anatomical characteristics of mantle in plan views (Fig.5. C & D) Mantle parenchymatous in all layers. Outer mantle layer: parenchymatous, (type L, Agerer)[START_REF] Agerer | Colour Atlas of Ectomycorrhizae (1st-11th del)[END_REF]; cells irregular in shape, 10 µm in diameter and 12.5 µm in length, light brown color of cells, no cell contents, no septa and clamps. Inner mantle layer: parenchymatous, (type M, Agerer)[START_REF] Agerer | Colour Atlas of Ectomycorrhizae (1st-11th del)[END_REF]; cells angular, smaller than outer mantle, 9 µm in diameter and 11 µm in length, honey brown color of cells, no matrix material, no septa and clamps. Anatomical characteristics of emanating elements (Fig.5. B). Rhizomorphs: differentiated (type B, Agerer)[START_REF] Agerer | Colour Atlas of Ectomycorrhizae (1st-11th del)[END_REF]; septa present and H-type anastomose type, cells 5 µm in diameter, 26 µm in length, cells thin walled, septa common, clamps and clamp septa absent.

Figure 5 .

 5 Figure 5. A-F. Ectomycorrhizae of Suillus sibiricus with Salixalba. View of ECM under stereomicroscope (A). Anatomical features of ECM under light microscope (B-D). Rhizomorph (B), Outer mantle (C), Inner mantle (D). Scale Bars for A = 1 mm, B = 17 µm, C = 8.5 µm, D = 16 µm.

Figure. 6 .

 6 Figure. 6. Bootstrap 50% majority-rule consensus tree. Cladogram based on parsimony analysis of rDNA-ITS region of different species of Suillus. MP tree generated by parsimony analysis of rDNA-ITS with 5.8s gene. The numbers above brackets refer to number of changes, those below to Bootstrap values. The accession numbers of analyzed sequences are sown after each taxon name.

  Proceedings of the 7 th International Conference on Mushroom Biology and Mushroom Products (ICMBMP7) 2011 In contrast to P. cembra, in present investigation S. sibiricus forms dichotomously branched mycorrhiza with P. wallichiana. The mycorrhizal system bears honey brown coloration with long spiny to cottony appearance. Mantel resembles with P. cembra mycorrhizal in its shape but cells varied from round to angular. The symbiotic range of S. sibiricus is revised in this investigation. A deciduous host, Salix alba L. is found symbiotically with S. sibiricus. It possesses monopodial pinnate type of mycorrhizal systems. These are different from P. wallichiana and P. cembra mycorrhiza. It posses parenchymatous mantle with irregular cells. Rare H-type rhizomorphs are present with dark brown-to black-colored septate hyphae.

	rhizomorphs.
	granulatus AY898617.1
	Section: Diversity and Taxonomy	132

Proceedings of the 7 th International Conference on Mushroom Biology and Mushroom Products (ICMBMP7) 2011

Section: Diversity and Taxonomy 130 basidiomata and ECM morphotypes of both mushrooms have been described and illustrated using morpho-anatomical methods. The S. sibiricus and its ECM mycorrhizae have been identified using rDNA-ITS and 5.8S gene. S. sibiricus was described as mycorrhizal from two different hosts viz; Pinus wallichiana and Salix alba while S. brevipes was found associated with Quercus incana.


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Monitoring viable airborne inoculum of Botrytis cinerea in the

Viable airborne inoculum of Botrytis cinerea was monitored bimonthly during 3 years (September 2007-December 2010) on a site in the South-East of France located approximately 5 km away from susceptible crops. Viable inoculum was observed on 96 % of the sampling days, including during cold winter periods and hot and dry summer conditions. The concentration of airborne inoculum was significantly higher during day-time than at night. Peaks of concentration were recorded at different periods each year (September-October in 2008, May in 2010). The abundance of viable inoculum was positively correlated with average daily relative humidity and negatively correlated with air temperature and solar radiation. The analysis of backward trajectories suggested that air masses originating from the North or the South brought more viable inoculum than those from the West. This study showed that susceptible crops may be at danger from viable inoculum of B. cinerea during all seasons of the year, but that risk prediction models could be developed on the basis of climatic conditions and the origin of air masses.

  South-East of France over 3 years: relation with climatic parameters and the origin of air masses Christel Leyronas, Philippe C. Nicot To cite this version: Christel Leyronas, Philippe C. Nicot. Monitoring viable airborne inoculum of Botrytis cinerea in the South-East of France over 3 years: relation with climatic parameters and the origin of air masses. 10. International Congress of Plant Pathology (ICPP), Aug 2013, Pekin, China. 620 p. hal-02745614 O01.006 Monitoring viable airborne inoculum of Botrytis cinerea in the South-East of France over 3 years: relation with climatic parameters and the origin of air masses C. Leyronas and P.C. Nicot INRA, UR407 Pathologie végétale, Domaine Saint Maurice,CS 60094, France Email: philippe.nicot@avignon.inra.fr