Pak. J. Bot., 44(6): 2093-2102, 2012. A NEW SPECIES OF TIAROSPORELLA AZADARICHTA AND NEW FUNGAL RECORDS ON AZADIRACHTA INDICA FROM PAKISTAN SYED QAISER ABBAS1, TEHREEMA IFTIKHAR1*, MUBASHIR NIAZ1, IFTIKHAR ALI1, NABILA IFTIKHAR1 AND ALIA ABBAS2 1 Department of Botany, Government College University, Alama Iqbal Road, Faisalabad, Pakistan 2 Department of Botany, Federal Urdu University, Karachi, Pakistan * Corresponding author’s e-mail: pakaim2001@yahoo.com Abstract A new species of Tiarosporella azadarichta has been described on Azadirachta indica and some new fungal records viz., Diplozythiella bambusina, Ulocladium chartarum, Cladosporium nigrellum, Cladosporium oxysporum. Didymostilbe coffeae, Muellerella pygmaea, Lasiodiplodia paraphysaria, Monochaetinula terminalae, Trimmatostroma sp., and Epidermophyton floccosum are reported on Azadirachta indica for the first time from Pakistan. Introduction Azadirachta indica (as Azadirachta Juss, Melia azadirachta) belong to the family Meliaceae. Neem is its common name. It is important due to its commercial and medicinal value. Azadirachta indica is also an important plant for its potential of anti-fungal, anti-bacterial and insecticidal activity. Decline of trees due to fungi are increasing tremendously in Pakistan especially in Punjab and Sindh (Javed et al., 2004; Khanzada et al., 2011; Fateh et al., 2011; Rheman et al., 2011; Farooq et al., 2011). Sixteen fungi has been reported on Azadirachta indica from Pakistan viz., Fuligo septica var. septica (L.) Wiggers on trunk of Azadirachta indica; Hypocreopsis macrostoma (Berk. & Curt.) Mueller on bark of Azadirachta indica (as Melia azadirachta); Pleospora pellita (Fr.) Rab, on stem of Melia azadirachta; Eutypa lundibunda Sacc.,on branches of Azadirachta indica (as Melia azadirachta); Eutypella russedes (Berk. & Br.) Berl, on dead branches of Azadirachta indica (Melia azadirachta); Ganoderma lucidum (Curtis) Karst. (as Fomes lucidus), on Azadirachta indica (Neem); Polyporus ostreiformis Berk., causes wood rot in Azadirachta indica (as Melia azadirachta); Fomes senex Nees et Mont., causes white wood rot of Azadirachta indica, Alternaria tenuissima (Nees. ex. Fr.) Wiltshire, causes leaf spot of Azadirachta indica; Cercospora subsessilis Syd., causes leaf spot of Azadirachta indica, Cercospora meliae Ell. & Ev., causes leaf spot on Azadirachta indica, Oidium spp., cause powdery mildew in Azadirachta indica; Botryodiplodia azedarachta (Ell. & Ev.) Ahmad; causes die back of Azadirachta indica; Lasiodiplodia theobromae (Pat.) Griffon & Maubl (as Botryodiplodia theobromae Pat., on dead branches of Azadirachta indica; Diplodia meliae Sacc. & Roum., on leaf rachis of Azadirachta indica; Dothiorella meliae Ahmad on leaf rachis of Azadirachta indica; Ahmad, 1956, 1962, 1969, 1978; Ahmad & Arshad, 1972; Ahmad et al., 1997; Ghaffar & Kafi, 1968; Ghaffar et al., 1971; Ghaffar & Abbas,1972; Ghafoor & Khan, 1976; Khan, 1952, 1969, 1989; Khan & Kamal 1963, 1968 Kamal & Mughal, 1968; Malik & Khan, 1944. It is very surprising that only 16 fungi have been reported from Pakistan on Azadirachta indica a widely cultivated and wildly grown tree. Why it is so? Whether this plant is neglected for fungal screening? or it is its antifungal, antibacterial and anti-insecticidal activity that play a role to protect this plant from pathogens. For this reason a detailed survey of the plant for fungi was under taken in this study. Materials and Methods Samples of Azadirachta indica were collected from the different areas of District Faisalabad and Gojra. The different areas were G.C. University. Faisalabad, Agriculture University Faisalabad, Gutwala forest (park) Faisalabad, Tandlianwala and Gojra City. Methods and materials are the same as described Abbas et al., (2010). Identification up to species level were carried out after consulting (Morris, 1963; Ellis, 1971, 1976; Carmichael et al., 1980; Sutton, 1980; Ahmad, 1978; Ahmad et al., 1997; Abbas et al., 2004; Kirk, 2012). Results and Discussion The fungus on Azadarichta indica specimen G.C.U.F. Mycol. H. # 40 was identified as a Diplozythiella bambusina Died. Diplozythiella bambusina Died., Annls mycol. 14: 215 (1916); Sutton, The Coelomycetes:58-61 (1980) Description of the fungus under study: Conidiomata eustromatic, ostiolate, dark brown to black, 152-190 µm. Conidiophores absent. Conidiogenous cells hyaline, enterogenous. Conidia uniseptate, hyaline, cylindrical, rounded at ends, constricted at septa, 3.8-11.4 × 1.261.50µm Fig. 1(A-E). Diplozythiella Died is a monotypic genus based on Diplozythiella bambusina Died. Fungus under study on Azadirachta indica was compared with the description of D. bambusina and found that the fungus under study completely resembled with D. bambusina, Therefore, the fungus on A. indica is identified as D. bambusina. Genus Diplozythiella has not been previously reported from Pakistan (Ahmad et al., 1997). It is an addition to fungal flora of Pakistan and Azadirachta indica is also a new host of Diplozythiella bambusina from Pakistan. Specimen examined: Diplozythiella bambusina on bark of Azadirachta indica; Pensara Road Gojra, Pakistan; 22 April, 07; G.C.U.F. Mycol. H. # 40: S. Qaiser Abbas & Nabila Iftikhar. 2094 The fungus on G.C.U.M.H.No.42 is described as Tiarosporella azadarichta sp. nov. Tiarosporella azadarichta sp. nov. Description of the fungus: Colony blackish grey appearance on natural sample. Conidiomata pycnidial, separate, globose, thick walled and dark brown, 150 µm in diameter, ostiole absent. Conidiogenous cells hyaline, (16.5-21 × 3.5µm). Conidia aseptate, cylindrical, hyaline 12.25-16.5 (mostly 14µm) × 3.5µm and covered with gelatinous sheath which form cap like structure on apical side of conidia Fig. 2(A-D). Latin description: Conidiomata pycnidiala, separatata, globosa, unilocularia 150µm dia., ostiola absentia, conidiophora non observa, cellulae conidiogenae hyalinae (16.5-21 × 3.5 µm). Conidia aseptata, hyalinae., cylindricae, mucilaginae enclosa, ad apicem capitata 12.25-16.5 (14µm) × 3.5 µm. Holotypus-Tiarosporella azadarichta in ramis emortius Azadarichta indica Green Town Gojra Pakistan; 3 May, 07; S. Qaiser Abbas & Nabila Iftikhar G.C.U.F. Mycol. H. # 42. Tiarosporella Höhn., and Tiarospora Sacc. & March are two genera, which closely resemble with the fungus found on Azadirachta indica. Resemblance lies In that both genera, have pycnidial conidiomata, dark brown, separate, globose, thick walled, of textura angularis and ostiole central, circular and papillate. Conidiophores absent. Conidiogenous cells hologenous and nonproliferating, discrete, lageniform, hyaline and smooth. However they differ from each other in some respect. Tiarosporella has aseptate conidia and Tiarospora has uniseptate conidia. The fungus under study on A. indica has aseptate, cylindrical, hyaline conidia with gelatinous sheath, therefore it belongs to the genus Tiarosporella. Tiarosporella azadarichta resembles with Tiarosporella graminis (Pirozynski & Shoemaker) Nag Raj, in having straight, cylindrical, ellipsoido-fusiform conidia with apical appendages but T. azadarichta differs from T. graminis (Pirozynski & Shoemaker) Nag Raj in size of conidiogenous cells and conidia. Conidiogenous cells of T. graminis are smaller and less wider (12-15×1.52.5µm) than T. azadarichta (16.5-21×3.5µm), whereas conidia in T. graminis are longer and wider (20-29.5×79µm) than T. azadarichta (12.25-16.5 (mostly 14×3.5µm). Similarly in T. graminis var. karoo conidiogenous cells are smaller and less wider (12-18×1.5-2.5µm) than T. azadarichta (16.5-21×3.5µm). Conidiomata of T. abietis Whitney, Reid & Pirozynski, T. parka (Berk. & Br.) Whitney, Reid & Pirozynski and T. pseudotsugae Whitening, Reid & Pirozynski are larger and wider (550600µm in diam.) than T. azadarichta (150-190µm in diam.). Furthermore in T. tritici Sutton & Marasas, conidiomata are slightly bigger and wider (200µm in diam.) than T. azadarichta whereas conidia in T. tritic are oval to fusiform, straight 29-38 x 12-17µm., more longer and wider conidia than T. azadarichta (16.5-21×3.5µm). T. madreeya (Subram. & Ramakr.) Nag Raj, closely resembled T. azadarichta in having slightly bigger conidiomata (200µm) than this fungus (150-190µm). SYED QAISER ABBAS ET AL., Similarly the conidiogenous cells of T. madreeya are more smaller and less wider (8-10×2.5-4µm) than T. azadarichta (16.5-21×3.5µm) and conidia are straight, cylindrical, much longer and wider (19-27×4.5-6µm) than T. azadarichta [12.25-16.5µm (mostly 14µm) × 3.5µm]. It is clear that T. madreeya is near to T. azadarichta, but conidia of T. azadarichta are smaller and less wider, therefore the under study fungus is described as a new species. Previously the genus Tiarosporella has not been reported from Pakistan (Ahmad et al., 1997). The genus Tiarosporella is a new addition to the fungal flora of Pakistan and Tiarosporella azadarichta is a new species described from Gojra, Pakistan. Specimen examined: Tiarosporella azadarichta from dead branch of Azadirachta indica; Green Town Gojra Pakistan; 3 May, 07; G.C.U.F. Mycol. H. # 42; S. Qaiser Abbas & Nabila Iftikhar. The fungus on Azadirachta indica specimen G.C.U.FMH. # 34 is identified as Ulocladium chartarum (Preuss) Simmons. Ulocladium chartarum (Preuss) Simmons, Mycologia, 59: 88-90 (1967). Description of the fungus under study: Colonies effuse and black. Mycelium, superficial, black brown. Conidiophores dark brown, branched, morphologically different from vegetative hyphae, 30-52.5×3.5-7µm. Conidiogenous cells olivaceous brown, cylindrical, 7×3.5µm. Conidia broadly ellipsoidal or inversely ovoid, brown 1-5 oblique and transverse septa, beak not present, thick walled, echinulated, 11.4-34.2×7.6-15.2µm Fig. 3(A-G). Genus Ulocladium and Alternaria show close resemblance with each other but differ in conidial attachment to conidiophores. In Alternaira conidia are attached from their broader side while in Ulocladium conidia are attached from their narrow side. Ulocladium chartarum (Preuss) Simmons, U. consortile (Thüm.) Simmons and U. alternarie (Cooke) Simmons are characterized in having 1-5 transverse septa and several longitudinal or oblique septa, whereas U. botrytis Preuss has 1-3 transverse speta. U. atrum Preuss., and U. oudamansii Simmons, have 3-5 transverse septa. U. clamydosporum Mouch has 2-7 transverse and several longitudinal and oblique septa. The fungus present on A. indica has 1-5 transverse septa and several longitudinal and oblique septa. These characters are common in 3 species of Ulocladium viz., U. chartarum, U. consortile and U. alternarie. However in U. chartarum (Preuss) Simmons conidiophores are 50 × 5-7µm, conidia (1838 × 11-20µm) in chains of 2-10 with small or false beaks. Whereas U. consortile and U. alternarie differ from U. chartarum in that conidia are without beaks and are not in chains (16-34 × 10-15µm). In U. consortile conidiophores are (60 × 4-5µm), whereas in U. alternarie conidiophores are 100 × 4-7µm and conidia are 18-35 × 15-20 µm, that is smaller than U. chartarum. In the fungus under study, conidial lower limit range 11.4-34.2 × 7.6-15.2µm is lower than U. alternarie 18-35 × 15-20µm and U. consortile 16-34 × 10-15µm and conidia are less wider than U. alternarie and U. consortile. NEW FUNGAL RECORDS ON AZADIRACHTA INDICA FROM PAKISTAN A C B 2095 D E Fig. 1(A-E). Diplozytheilla bambusina: A, B, C & D. Conidia.1000X; E. conidiomata.400X. B A D C Fig. 2(A-D). Tiarosporella azadarichta A. Conidia with mucilaginous cap. 1000X; B. Conidia with gelatinous sheath.1000X; C. Conidia.1000X; D. Conidiomata.400X. A D B E C F G Fig. 3(A-G). Ulocladium chartarum: A. Conidiophores with conidial attachment; B. Conidiophore with unseptate conidia (400X); C. Conidiophore (1000X). D. Attachment of conidia; E. Chain of 3 conidia; F. Chain of 2 conidia; G. Conidia with variable septa number (D, F, G, 1000X; E, 400X). The fungus under study completely resembled with Ulocladium chartarum except some minor differences in conidial dimension, therefore, it is identified as Ulocladium chartarum. Three Ulocladium spp., viz., Ulocladium botrytis Preuss, U. atrum Preuss., U. consortiale (Thüm.) Simmons, have been reported from Pakistan (Ahmad, 1969; Nisa et al., 1971; Matsushima, 1993). U. chartarum has not been previously reported from Pakistan (Ahmad et al., 1997). U. chartarum is an addition to fungal flora of Pakistan and Azadirachta indica is a new host record of U. chartarum. Specimen examined: Ulocladium chartarum Preuss, on branches of A. indica; Green town Gojra Pakistan; 29 April, 07; G.C.U.F. M.H. # 34; S.Q. Abbas and Nabila Iftikhar. 2096 The fungus on Azadirachta indica specimen G.C.U.FMH. # 37 is identified as Cladosporium nigrellum Ellis & Everh. Cladosporium nigrellum Ellis & Everh., Proc. Acad. N. Sci. Phalid. 1893: 463 (1894). Description of the fungus under study: Mycelium septate, superficial, pale brown. Conidiophores septate, brown, scars present 230-250×5-9µm. Conidia cylindrical to oval, attached at apex as well as at lateral sides of conidiophores, 0-3 septate, 11.4-16 × 3.8-8µm Fig. 4(A-E). The fungus under study on Azadirachta. indica has 03 septate conidia. Cladosporium nigrellum Ellis & Everh, C. apicale Berk.& Brown, C. uredinicola Speg., C. macrocarpum Preuss, C. variable (Cooke) de Vries, and C. brassicae (Ellis & Barthol.) Ellis have 0-3 septate conidia. Inspite of similarity in having 0-3 septate conidia, howevere there are sufficient differences among them are found, which separate them from one another. Cladosporium brassicae have shorter conidiophores (150×6-9µm) with terminal and intercalary swellings of 10-12µm. than fungus under study where conidiophores are 230-250×5-9µm; and without terminal and intercalary swelling. Similarly C. apicale also have wider conidiophores 260×6-8µm than fungus under study 230250×5-9µm whereas C. variable have long conidiophores 350×6-8µm than fungus under study which has conidiophores of 230-250×5-9µm and Cladosporium macrocarpum differs from fungus under study in having longer and less wider conidiophores 300×4-8µm with terminal and intercalary swellings of 9-11µm diameter. Furthermore Cladosporium uredinicola also differs from under study fungus in having longer and less wider conidiophores 300×3-5µm than the under study fungus where conidiophores are 230-250×5-9µm. Furthermore conidiophores in C. nigrellum are wavy, smooth, reddish brown, septate, 250 × 5-9µm; Conidia cylindrical, narrowing at the ends, lemon shaped, in simple or branched chains, smooth, light brown, 5-15×4-7µm. After comparison it is concluded that the under study fungus on Azadirachta indica closely resembled with C. nigrellum, hence it is identified as C. nigrellum. The fungus on A. indica identified as C. nigrellum has not been previously reported from Pakistan (Ahmad et al., 1997) and it is a new report on A. indica from Gojra Pakistan. SYED QAISER ABBAS ET AL., chains. Chains consist of 3-4 conidia (Fig. 4). Colony on Azadirachta indica has green appearance Fig. 5(A-E). Cladosporium orchidearum Cooke & Massee, C. brassicae (Ellis & Barthol.) Ellis, C. uredinicola Speg., C. nigrellum Ellis & Everh., C. macrocarpum Preuss and C. variable (Cooke) de Vries have 0-1 septate conidia and differ from under study fungus which has 0-3 septate conidia. However, Cladosporium oxysporum Berk. & Curt., C. herbarum (Pers.) Link ex Gray, C. colocasiae Sawada, C. orchids E. A. Ellis & M. B. Ellis, C. gallicola Sutton, C. aecidiicola Thüm, and Cladosporium tenuissimum Cooke have 0-1 septate conidia and resemble with under study fungus in this regard, however there are some differences which separate each from other Cladosporium gallicola has longer and wider conidiophores (250×5-9µm) than under study fungus (152-268×3.8µm). Similarly C. aecidiicola (100×4-6µm) and Cladosporium orchids (100×3-8µm) have shorter and slightly wider conidiophores than under study fungus (152-268×3.8µm). Further more C. tenuissimum has much longer conidiophores (800×3-6µm) than under study fungus (152-268×3.8µm) and C. herbarum has conidia, smaller and wider (5-23×3-8µm) than under study fungus (10-19×3.8-5.7µm). Fungus under study completely resembles with Cladosporium oxysporum where conidiophores can easily be differentiated from vegetative hyphae, straight or slightly wavy, swollen nodes near the apex and upto 500 µm long and 3-5 µm wide with terminal and intercalary swellings (6-8 µm). Conidia arising from terminal swellings which later become smooth, intercalary, forming short simple and branched chains, (5-30×3-6µm). Fungus on Azadirachta indica shows its complete resemblance with C. oxysporum, therefore it is identified as C. oxysporum. Previously Cladosporium oxysporum Berk. & Curt., has been also recorded on 25 different plants belonging to different families from Pakistan (Ahmad et al., 1997). However it is first time recorded on Azadirachta indica from Gojra Pakistan. Specimen examined: Cladosporium oxysporum on leaves of Azadirachta indica; Quaid.e.Azam School, Gojra, Pakistan; 19 September, 07; G.C.U.F. Mycol. H. # 36 S. Qaiser Abbas & Nabila Iftikhar. The fungus on Azadirachta indica specimen G.C.U.FMH. # 38 is identified as Didymostilbe coffeae Henn. Didymostilbe coffeae Henn. Hedwigia, 41: 148 (1902). Specimen examined: Cladosporium nigrellum from leaves of Azadirachta indica; Green Town Gojra Pakistan; 10 September, 07; G.C.U.F. Mycol. H. # 37; S. Qaiser Abbas & Nabila Iftikhar. The fungus on Azadirachta indica specimen G.C.U.FMH. # 36 is identified as Cladosporium oxysporum Berk. & Curt Cladosporium oxysporum Berk. & Curt., (1868); Ellis. More Dematiaceous Hyphomycetes, CAB(IMI), Kew, Surrey, England, pp. 507, (1976). Description of the fungus under study: Mycelium superficial, light brown, septate. Conidiophore olivaceous brown, macronematous, 152-268×3.8µm, chlamydospores present, 7.6 µm. Conidia sub rounded, ellipsoidal to cylindrical, 0-1 septate, 10-19×3.8-5.7µm, solitary or in Description of the fungus under study: Mycelium septate, branched. Synnemata capitate, 816×38µm, apex broad, 49.468.4µm; hyaline, thick and steriated. Conidiophores 1927×3.8µm. Conidia uniseptate, obovoid to cylindrical, apex and base obtuse 4.6-6.5×3.04-3.8µm. Fig. 6(A-G). Under study fungus found on Azadirachta indica resembles with genera Didymobotryum Sacc., and Didymostilbe Hennings. Both genera have tall cylindrical stipe. Conidia uniseptate, oblong or cylindrical but the difference between the two genera is that in Didymobotryum conidia are dry and in Didymostilbe conidia were produced in mucoid mass.The fungus isolated from Azadirachta indica (Neem) closely resemble with genus Didymostilbe. The fungus from A. indica was identified as Didymostilbe coffeae Henn., after consulting Morris, (1963); Ellis (1971, 1976). NEW FUNGAL RECORDS ON AZADIRACHTA INDICA FROM PAKISTAN A B C 2097 D E Fig. 4(A-C). Cladosporium nigrellum: A. uniseptate conidia with prominent scars; B. uniseptate conidia; C. biseptate conidia; (A, B, C, 1000X). D & E. Conidial attachment (D & E, 400X). B A C D E Fig. 5. Cladosporium oxysporum. A, B & C. aseptate & uniseptate conidia (A, B, C 1000X). D & F. Conidiophores with conidial attachment (D400X; E1000X). Previously Didymostilbe coprophila has been reported on dung from Faisalabad Pakistan (Mirza & Qureshi, 1970). D. coffeae is a new fungal record from Pakistan and Azadirachta indica is a new host for this fungus from Faisalabad, Pakistan. Specimen examined: D. coffeae from bark of Azadirachta indica; Tandlianwala district Faisalabad Pakistan; 15 September, 07; G.C.U.F. Mycol. H. # 38; S.Q. Abbas & Nabila Iftikhar. Fungus on Azadirachta indica specimen G.C.U.FMH. # 39 is identified as Muellerella pygmaea (Körb.) D. Hawksworth Muellerella pygmaea (Körb.) D. Hawksworth. Bot. Notiser, 132(3): 289 (1979) =Eendococcus pygmaeus (Körb.) Th. Fr. =Microthelia ecatonospora Anzi, Atti Soc. ital. Sci. nat. (Modena), 9: 256 (1866) =Microthelia pygmaea (Körb.) Körb., (1855) =Muellerella pygmaea (Körb.) D. Hawksw., Bot. Notiser, 132(3): 289 (1979) var. pygmaea =Mcoporum pygmaeum (Körb.) Jatta, (1900) =Pyrenula pygmaea (Körb.) Tuck., Gen. lich. (Amherst): 272 (1872) =Sychnogonia pygmaea (Körb.) Trevis., (1860) =Tichothecium pygmaeum Körb., Parerga lichenol. (Breslau): 467 (1865); Ahmad, Ascomycetes of Pakistan, part 1. Biological society of Pakistan, monograph 137(1978). =Tichothecium pygmaeum var. ecatonosporum Anzi & G. Winter (1885) =Tichothecium pygmaeum Körb., Parerga lichenol. (Breslau): 467 (1865) var. pygmaeum Description of the fungus under study: Ascocarp flaskshaped, dark brown, superficial, ostiolate with colorless appendages on ostioler region, 425.5-485.5µm. Asci clavate, 28-35×10.5-14µm. Ascospores hyaline, many, oval to ellipsoidal, uniseptate, 7×3.5µm. Fig. 7(A-E). This fungus Muellerella pygmaea (Körb.) D. Hawksworth was reported by Ahmad (1978) and Ahmad et al., (1997) as Tichothecium. Pygmaeum from Changa Manga Pakistan on Salvadora oleoides. Hawksworth (1979) replaced it to Muellerella pygmaea (Körb.) Hawksworth. Fungus under study completely resembled with Muellerella pygmaea (Körb.) Hawksw (syn.T. pygmaeum Köerb) in having ascocarp of flask-shaped, dark brown, superficial and ostiolate, 425.5-485.5 µm; appendages hyaline at ostioler region. Asci clavate, 28-35×10.5-14µm. Ascospores hyaline, many, uniseptate, oval to ellipsoidal, 7×3.5µm Azadirachta indica is a new host for Muellerella pygmaea (Körb.) D. Hawksw from Gojra Pakistan. Specimen examined: Muellerella pygmaea (Körb.) D. Hawksw as Tichothecium pygmaeum) on bark of Azadirachta indica; Bilal park Gojra Pakistan; 20 April, 07; S.Q. Abbas & Nabila Iftikhar. G.C.U.F. Mycol. H. # 39. SYED QAISER ABBAS ET AL., 2098 B A D E C F G Fig. 6(A-G). Didymostibe coffeae: A synnemata (60X); B,C capitates head of synnemata 9400X); D,E,F,G uniseptate conidia (1000X). A B A C B D E Fig. 7. Muellerella pygmaea: A. Ascocarp 400 X; B. Ascocarp with asci 400 X; C. Crushed ascocarp. 400X; D. Ascospores. (1000X). E. Asci.1000X. C Fig. 8(A-C). Lasiodiplodia paraphysaria: A. Hyaline conidia. 1000X; B. Mature conidia.1000X; C. Conidia with conidiogenous cells.1000X. NEW FUNGAL RECORDS ON AZADIRACHTA INDICA FROM PAKISTAN Fungus found on Azadiarchta indica specimen GCUF#42 is identified as Lasiodiplodia paraphysaria (Sacc) Keissl. Lasiodiplodia paraphysaria (Sacc.) Keissl., Beih. bot. Zbl., Abt. 2 36: 314 (1918) =Diplodia paraphysaria Sacc., Bull. Soc. R. Bot. Belg., 35: 130 (1896) Description of the fungus under study: Brownish black patches on the bark of Azadirachta indica. Conidiomata pycnidial, non ostiolate, dark brown, thick-walled, 296 µm. Conidiophore absent. Conidiogenous cells hyaline, unseptate, thin walled, 9-9.5×3.8µm. immature conidia hyaline, aseptate, oval and thickwalled, at maturity conidia become brown, uni-septate, thick walled with longitudinal striations, from apex to base 30-33.4×1416µm Fig. 8(A-C). Sutton (1980) was of the opinion that correct name of Botryodiplodia theobrome was Lasiodiplodia theobromae. Name of species L. abnormis L. citricola L. crassispora L. fiorii L. gilanensis L. gonubiensis L. hormozganensis L. iraniensis L. margaritacea L. paraphysaria Fungus under study L. parva L. plurivora L. pseudotheobromae L. ricinii L. rubropurpurea L. theobromae L. thomasiana L. undulate L. venezuelensis Howevere, Punithalingum (1980) dealt it as Botryodiplodia theobrome in his monograph. Abbas et al., (2004) when reassessing the Sphaeropsis undulata, they pointed out that Sphaeropsis undulata Berk & Curt., was an earliar name for Lasiodiplodia theobromae. After 2004, work on Lasiodiplodia was carried out both morphological as well on DNA finger printing and sequence (Pavlic et al., 2004; Burgess et al., 2006; Damm et al., 2007; Alves et al., 2008); Abdollahzadeh et al., (2010) dealt 14 species of Lasiodiplodia. Abdollahzadeh et al., (2010) was of the opinion that conidial dimension of Botryodiplodia theobrome never exceed 30 µm in length and 16 µm in width, while the conidial length in Lasiodiplodia undulata are up to 32µm and width is up to 19.2µm therefore they consider that both species are separate taxa. Total species of Lasiodiplodia spp., are tabulated with reference to conidial measurement (Abdollahzadeh et al., 2010). Conidial measurement (µm) 25-28 × 13-15 22.5-26.6 × 13.6-17.2 27-30 × 14-17 24-26 × 12-15 28.6-33.4 × 15.6-17.6 32-36 × 16-18.5 19.6-23.4 × 11.7-13.3 18.7-22.7 × 12.1-13.9 14-17 × 11-12 30-32 × 15-16 30-33.4 × 14-16 µm 18.3-22.1 × 10.7-12.3 26.7-32.5 × 14.4-16.7 25.5-30.5 × 14.8-17.2 21.7-26.3 × 13.4-14.8 16-19 × 10-11 24-33 × 13-17 23.6-28.8 × 13-15.4 22.4-24.2 × 12.9-14.3 28-30 × 11-12 20-32 × 13.5-19.2 26-33 × 12-15 Fungus under study differs from the following Lasiodiplodia spp., in having bigger conoidia viz., L. abnormis (25-28×13-15 µm), L. citricola (22.5-26.6×13.617.2 µm), L. crassispora (27-30 × 14-17 µm), L. fioriii (2426 × 12-15 µm), L. hormozganensis (19.6-23.4 × 11.7-13.3 µm), L. iraniensis (18.7-22.7 × 12.1-13.9 µm), L. margaritacea (14-17 × 11-12 µm), L. parva (18.3-22.1 × 10.7-12.3 µm), L. pseudotheobromae [(25.5–30.5× 14.8– 17.2 µm, Alves et al., 2008). (21.7-26.3 × 13.4-14.8, Abdollahzadeh et al., 2010)], L. ricinii (16-19 × 10-11 µm); L. theobromae[ ( 23.6-28.8 × 13-15.4 µm; Alves et al., 2008), (22.4-24.2 × 12.9-14.3 µm, Abdollahzadeh et al., 2010)]. Howevere L. gonubiensis (32-36 × 16-18.5 µm) has more longer and wider conidia than the fungus under study. L. gilanensis (28.6-33.4 × 15.6-17.6 µm ), L. thomasiana (28-30 × 11-12 µm) and L. undulata (20-32 × 13.5-19.2 µm) differ from the fungus under study by having smaller conidia, since their upper limit of conidial length is near or less than the lower length range of fungus under study. 2099 Reference Saccardo (1913) Abdollahzadeh et al., (2010) Burgess et al., (2006) Saccardo (1913) Abdollahzadeh et al., (2010) Pavlic et al., 2004 Abdollahzadeh et al., (2010) Abdollahzadeh et al., (2010) Pavlic et al., (2008) Saccardo (1913) Present study Alves et al., (2008) Damm et al., (2007) Alves et al., (2008) Abdollahzadeh et al., (2010) Saccardo (1913) Burgess et al., (2006) Alves et al., (2008) Abdollahzadeh et al., (2010) Saccardo (1913) Abbas et al., (2004) Burgess et al., (2006) Further more conidial width of L. undulate is more wider than the conidial width of fungus under study. Fungus under study (30-33.4 × 14-16µm) more closely resembles with L. paraphysaria (30-32 × 15-16 µm), therefore it is identified as Lasiodiplodia paraphysaria Lasiodiplodia paraphysaria is for the first time reported from Gojra, Pakistan. Specimen examined: Lasiodiplodia paraphysariais on bark of Azadirachta indica; 305 J.B. Gojra, Pakistan; 10 April, 07; S. Qaiser Abbas & Nabila Iftikhar G.C.U.F. Mycol.H. # 42. Fungus found on Azadiracta indica specimen # G.C.U.F. Mycol. H. # 44is identified as Monochaetinula terminalae (Bat. & Bezerra) Muthumary, Abbas & Sutton. Monochaetinula terminalae (Bat. & Bezerra) Muthumary, Abbas & Sutton, Trans. Br. Mycol. Soc., 87(1): 103-108, (1986). SYED QAISER ABBAS ET AL., 2100 Description of the fungus under study: Conidiomata eustromatic, globose, dark brown with rough surface, 114µm. Conidiogenous cells hyaline, cylindrical, hologenous, 5-7 × 2µm. Conidia 3-euseptate, ellipsoidal, hyaline, simple appendages or beak on both sides, 20.926.6 × 3.8µm. The length of basal appendage 2-2.85µm, the length of terminal appendage 5.7-7.6µm. Fig. 9(A-C). Genus Monochaetia (Sacc.) Allesch. (1902) and Monochaetinula Muthumary, Abbas & Sutton (1986) show close resemblance with under study fungus. and have already been reported from Pakistan. Genus Monochaetia (Sacc.) Allesch. is characterized by acervular conidiomata and 4euseptate conidia. While Monochaetinula Muthumary et al., (1986) has eustromatic conidiomata and 3-euseptate conidia. The under study fungus completely matches with Monochaetinula terminalae in having similarity in conidiomata, conidiogenous cells, conidiophores and conidial measurements. Therefore the fungus under study was identified as Monochaetinula terminaliae. Previously Monochaetinula terminaliae was reported from Pakistan on stem of Capparis decidua and Mimosa hamata, Muthumary et al., (1986). Azadirachta indica is an addition to host list of Monochaetinula terminaliae from Faisalabad Pakistan. Specimen examined Monochaetinula terminaliae on bark of Azadirachta indica; G.C.University Faisalabad Pakistan; 26 April, 07; S. Qaiser Abbas & Nabila Iftikhar G.C.U.F. Mycol. H. # 44. A A B C B C D E Fig. 9(A-C). Monochaetinula terminalae: A. Pycnidiom.100X; B. Conidium.1000X; C. Conidia with conidiogenous cells.1000X. Fig. 10(A-E). Trimmatostroma sp. A. Conidiogenous cell; B. uniseptate conidium; C. biseptate conidium. (A, B, C, 1000X), D. Conidial attachment; E. Conidia. (D & E, 1000X). Fungus found on Azadirecta indica specimen # G.C.U.F. simple and branched chains of variable in shape with I several transverse and longitudinal or oblique septa. The size of conidia of Trimmatostroma scutellare (Berk. & Br.) Ellis., T. eriodyctyonis (Dearn. & Barthol.) Ellis., and T. macowanii (Sacc.) Ellis also differ from the conidia of the fungus under study in having smaller conidia. Conidia are 10-30×8-25µm in T. scutellare ; 1450×7-26µm in T. eriodictyonis and 8-20×5-9µm in T. macowanii, whereas conidia of under study fungus are 45.6-182.4×7.6-15.2µm. Furthermore T. salicis and T. betulinum have solitary conidium, often forming fork like structure, clavate and smooth or slightly verrucose and obtuse at the ends but both species differ in conidial size. In T. salicis conidia are 12-38×4-10µm and conidia in T. betulinum, are 5-20×5-14µm. Therefore it is concluded that the species under study does not match with any Mycol. H. # 35 is identified as Trimmatostroma sp. Description of the fungus under study: Colonies dark brownish black on natural substrate. Stroma large, thick and brown. Conidiophore micromenatou slightly different from vegetative hyphae, unbranched, straight or wavy, pale brown and verrucose, 21-23×6-7µm. Conidiogenous cells terminal and cylindrical, 9-10×7µm. Conidia thick walled, highly variable in size and shape, 1-several transverse and oblique septa, brown, 45.6-182.4×7.615.2µm. Fig. 10(A-E). Trimmatostroma scutellare (Berk. & Br.) Ellis., T. eriodyctyonis (Dearn. & Barthol.) Ellis., and T. macowanii (Sacc.) Ellis., differ from T. salicis Corda, and T. betulinum (Corda) Hughes by forming conidia in NEW FUNGAL RECORDS ON AZADIRACHTA INDICA FROM PAKISTAN species of Trimmatostrom and looks a new species, will be published in some where else. Previously Trimmatostroma betulinum (Corda) Hughes., has been reported on dead branches of unknown host; Ahmad (1977) and Trimmatostroma myrti (Lind.) Hughes on dead branches of un-known host; Ahmad (1977) and from Khanspur Streams, Dadar Streams from Pakistan (Iqbal & Bhutty, 1979, 1980; Iqbal & Shahbaz, 1990). Azadirachta indica is a new host record of Trimmatostroma sp., from Faisalabad Pakistan. 2101 However the fungus found on G.C.U.F. Mycol. H. # 43. identified as Epidermophyton flocossum on Neem tree is first report in this connection from Faisalabad, Pakistan. Specimen examined Epidermophyton floccosum on bark of Azadirachta indica; G.C. University Campus Alama Iqbal Road, Faisalabad, Pakistan; 27 April, 07; G.C.U.F. Mycol. H. # 43; S. Qaiser Abbass & Nabila Iftikhar. Specimen examined Trimmatostroma sp., on branch of Azadirachta indica; Samanabad Gojra Pakistan; 25 April, 07; G.C.U.F. Mycol.H. # 35; S.Q. Abbas & Nabila Iftikhar.. Fungus found on Azadirecta indica specimen # G.C.U.F. Mycol. H. # 43. Epidermophyton flocossum (Harz) Langeron et Miloch Epidermophyton flocossum (Harz) Langeron et Miloch., (1930); Beneke, E. S. Medical mycology laboratory manual, pp. 66, (1962). Description of the fungus under study: Colonies white in color when grow on natural substratum. Mycelium superficial, thin, long and branched. Conidiophores hyaline and branched. Macro-conidia found, 3 septate, clavate, 10.5-17.5×3.5-7µm. No microconidia are found. Microsporium Velen., and Epidermophyton Sabourd are two closely related hyphomycetous genera, resemble each other in many aspects. Both have velvety colonies, grow rapidly on medium with tan to brown in color. Hyphae septate, large. Macroconidia hyaline, multicellullar, transversely septate, thick or thin walled. However they also differ from each other. In genus Microsporium, microconidia may appear on short hyphae. Macroconidia fusiform, often have an annular frill. Chlamydospores may present a fungal colony of buff to brown in color while in Epidermophyton colony color changes from buff to white at maturity. Macroonidia smooth and clavate-shaped with obtuse ends. They are found singly or in clusters, Fig. 11(A-C). Characteristics of under study fungus completely matched with Epidermophyton flocossum (Harz) Langeron & Miloch. Epidermophyton floccosum is generally human pathogen recorded from the world (Kazmi, 2004, Bundu & Pavihran 2002; Macit, 2005; Mohmoudabadi, 1997 and from Pakistan (Khan & Anwar, 1969, Dilnawaz & Naseer, 2001; Hussain et al., 1994; Thebo et al., 2006). Recently a project on Dermatophytes of District Faisalabad was carried out collaboration of Department of Botany, G.C. University Faisalabad and Department of Dermatology. HO Faisalabd where one hundred and seventy nine patients were studied for dermatophytic infection. In 11 patients Epidermophyton flocossum was isolated (7-13%). This is a high % of occurrence of it a there is a big question that how this pathogen perpetuate in this area? Is this soil borne or borne in other substrate? The isolation from Neem tree is further alaraming to the mycologist working on plant pathogenic fungi. Generally it is supposed that they are non pathogenic to animals and human being. A B C Fig. 11(A-C). 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