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Int. J. Biosci.2014International Journal of Biosciences IJB ISSN: 2220-6655 (Print) 2222-5234 (Online)http://www.innspub.netVol. 4, No. 1, p. 143-148, 2014RESEARCH PAPEROPEN ACCESSReport primary of Fusarium Proliferatum and Fusarium solaniagents of Dianthus caryophyllus wilting in Markazi province inIranB. Fattahi1, H. Rahanandeh2*, S. Chavoshi3, H.R. Zamanizadeh4, H. Bayat5, M.Moshayedi61Department of Plant Protection, Research & Science Branch, Islamic Azad University, Iran2Member of Department of Agronomy and Plant Breeding, Faculty of Agriculture, Rasht IslamicAzad University P. O. Box 4415866865, Lahijan, Iran3Depaetment of Agronomy and Plant Breeding, Faculty of agriculture, Arak Islamic AzadUniversity, Iran4Research & Science Branch, Islamic Azad University, Iran5National Ornamental Plants Research Station,Mahallat, Iran6Young Researchers Club of Rasht, Rasht Islamic Azad University, IranKey words: F. Proliferatum, F. solani, carnation, crown rot.doi: http://dx.doi.org/10.12692/ijb/4.1.143-148Article published on January 01, 2014AbstractWilt disease and root and crown rot important diseases of carnation in carnation greenhouses in Markaziprovince of Iran. In this study, was reported major cause of the disease, Fusarium species. Fusarium speciesassociated with different parts of growing carnations isolated in 2010 - 2012 years in Markazi province. 68isolates of Fusarium species Identification were based on colony morphology and conidial and perithecialcharacteristics, F. oxysporum, F. proliferatum, F. solani, F. equiseti. To our knowledge, this is the first report ofa wilt disease caused by F. proliferatum and F. solani on carnation in Iran.Populations of species wasRespectively 25 and 17.64 percent. Experimental results demonstrate that the virulence species F. proliferatumis more than F. solani.* CorrespondingAuthor: H. Rahanandeh rahanandeh20@yahoo.com143 Fatahi et al.
Int. J. Biosci.2014IntroductionThe purpose of this research is to identify and isolateCarnation (Dianthus caryophyllus L.) is one of thenew strains of Fusarium wilt carnation cause of Iran.most important commercially grown flowers of theworld (Anon., 2004). The perpetual floweringMaterial and methodcarnation, Dianthus caryophyllusL., is one of tenSampling and assessmentmajor cut-flower crops in Iran.A fixed plot survey was taken up during 2010-12, toknow the incidence of diseases, occurring onFusarium wilts, caused by F. oxysporum is one of theornamental plants grown under protected cultivationmost widespread and destructive diseases of manyin Markazi Province in Iran districts. The diseasemajor ornamental and horticultural crops.incidence was assessed by recording the number ofplants showing disease symptoms and the totalAll over the world in carnation culture, vascular wiltnumber of plants present. In each greenhouse, rowscaused by Fusarium oxysporum Schelchtend.:Fr. f.were selected randomly and the number of plantssp. dianthi (Prill. & Delacr.) W.C. Snyder & H.N.showing typical symptoms and the total number ofHans, is the most important fungal disease. Fusariumplants were recorded. Per cent disease incidence wasoxysporum f. sp. dianthi and F. avenaceum (Fr.)calculated by using the formula given by wheelerSacc. were consistently isolated from wilted carnation(1969).plants in commercial stocks in south-west England(Carver et al., 1996). These are the entities which takePer cent disease incidence No. of plants showingheavy toll of plants and cause economic losses to thewilting symptoms x 100 Total no. of plants observedgrowers, once established in a greenhouse. Since,During survey, characteristic symptoms of thegreenhouse conditions which favor luxuriant growthdiseases were recorded and also samples wereof crops, also favor’s the development of plantcollected for isolation of pathogens.pathogens Garibaldi et al. (2004) , isolated Fusariumspp. consistently and readily from symptomaticMediumvascular tissue onto a Fusarium-selective mediumPurification and identification of the pathogen wasfrom the wilt affected plants. Colonies were identifiedcultured, PDA and CLA. Key on the mycologicalas F. oxysporum after sub-culturing on potatoNelson et al., (1983) evaluated growth and generativedextrosestructures, the fungal isolates were ns. The first symptom of wilt of carnationis yellowing of leaves, followed by withering of leafIsolation of pathogensbases and yellowing of midribs, which progressesThe infected plant parts were surface sterilized withfrom the base of the leaf. The infected leaves1:1000 mercuric chloride (HgCl2) solution for 30gradually become chlorotic and finally wilt. Someseconds and washed separately in sterilized distilledtimes,water twice to remove the traces of mercury, if anyunilateraldevelopmentofsymptomsproducing crook-neck can also be seen (Sohi, 1992).Inand2011 the first report of a wilt disease caused bycontainingF. proliferatum on carnation in China (Zhang et al.,Petriplates were incubated at room temperature 250C2011). Fusarium important factor causing damageand observed periodically for the growth of pureto the carnation in Iran. Fusarium damage incolonies. The pure colonies which developed from thegreenhouses up to 60%. Fusarium oxysporum speciesbits were transferred to PDA slants and incubated athave been reported from Iran.250C for 15 days. Then such slants were used to Petriplates(PDA).Thethe cultural characters in the laboratory (Ben-Yephetet al., 1994).144 Fatahi et al.
Int. J. Biosci.2014Identification of FusariumProving pathogenicity byThis method was followed for maintaining pure(plants) with a spore suspensioncultures.Rooted cuttings (plugs) of carnation was planted in aDilutesporesuspensions(8-10inoculating the soilmacroconidia /ml) of the isolated pathogens (F.steam sterilized potting media consisting of soil, sandProliferatum and F. solani) were prepared in sterileand farm yard manure in the ratio of 3:1:1. Further,distilled water. One ml of such suspension was spreadthe sick soil was made by inoculating the giant cultureuniformly on two per cent water agar plates and theof F.proliferatum and F.solani to the sterile soilexcess of which was aseptically drained. Such platesrespectively. A control treatment was maintainedwere incubated at 250C and periodically observed forwithout adding the inoculum. Observations weregermination of spores under the microscope. Hyphaemade regularly for the appearance and developmentcoming from each end cell of the single spore wasof symptoms. After symptom development, re-traced and marked with the ink on the reverse side ofisolation was done from the artificially infectedpetriplates. Then tip of hypha was cut and transferredplants. The isolate obtained was compared with theto PDA slants with the help of cork borer underoriginal culture for confirmation (Sharma, 2000).aseptic conditions and incubated at temperature of 25C for 10 days. Later, mycelial bits of the fungus wereResultplaced in the center of Petriplates containing potatoDuring 2010-2012, this study was conducted indextrose agar medium and incubated at 25 C for 10greenhouse carnation in province markazi. Samplesdays. No saltation or sectoring was observed in thewere transported to the laboratory for isolation ofculture and it was concluded that, it was a purepathogen.68 cases were related to the genusculture of the fungus. The spores of the pathogensFusarium, 28 sample genus Phtophthora ,25 Numberwere taken from infected plant portions (collarPythium,region) and temporary slide mounts were prepared inRhizoctonia. Greenhouses that showed symptoms oflactophenol. Then they were observed under highinfection, the fungus was isolated from five ernaria,23samplesporesgenera. Abundance fungi are shown in Table 1. Based(macroconidia, microconidia and chlamydospores) ofon the color specifications of colony growth rates ofthe pathogens were observed under microscope andisolates, microscopic characteristics of four species ofmeasured using ocular and stage micrometer (NelsonFusarium were identified using the keys of Nelson etet al., 1972).al., 1983. Two species, F. Proliferatum and F. solani,first reported of carnation in Iran. Abundance speciesProving pathogenicity by inoculating Dipping thewere studied in 21.79 and 15.38 percent.roots in a spore suspensionRootswasplacedconcentration of106inasporesuspensiontofor 25 minutes. The seedlingsTable 1. The number of isolates obtained from theinitial sampling.were grown in pots with a diameter of 14 cm. ThenIsolated fungiNumber of samplesseedlings in pots with a diameter of 14 cm wereFusarium spp.68cultured pasteurized soil. The roots of controlPhytophthora sp.28seedlings were placed in water. Then seedlings in potsPythium sp.25Rhizoctonia sp.23Alternaria sp.22with a diameter of 14 cm were cultured pasteurizedsoil The pots were kept for 1 month in the greenhouseconditions. Of plants discolored, yellow and faded(leaf chlorosis symptoms) (percent wilting) wererecorded (Carver et al., 1996).Fusarium proliferatum var proliferatumColony characteristics Fusarium proliferatum varproliferatum growth on PDA medium at 25 C after 10days of 8 cm. Color of colonies on the surface of PDA145 Fatahi et al.
Int. J. Biosci.2014medium, cream, purple, turning to purple to darkColony characteristics Fusarium solani var solanipurple ranged. Relatively abundant aerial hyphae andPlated on potato dextrose agar and incubated at 25 C.cotton first medium purple and dark purple to white,After ten days, individual fungal colonies werethen changes color. Give spores. After 2 days ofsubcultured on to PDA plates for identification.culturing the fungus was produces spores on PDAWithin 10 days, Cream to light violet, aerial myceliumand CLA Located on the side of phyalides micro aerialdeveloped. With the aid of an inverted microscope,conidia production is only, but then the macrosingle conidia were transferred to carnation leaf agarproduces conidia. May be produced after 2 weeks(CLA) medium. after 2-3 days were produced 10daysofincubation,morphologicalconidiogenesis cell monophialide and polyphialidecharacteristics were found to be identical to those ofbranchedF. solani. On CLA medium, conidia grew in branchedandNon-branching.Aerialhyphaeproduced laterally primary conidiophore. nched conidiophore and then will branchingmonophialides or polyphialides. No conidiospores inBut secondary conidiophore are located in densechains were observed. Microconidia were ovate tosporodochium (Fig. 1).Frequency and microconidialong and oval, 0 to 1 septate, and 9 to 11 1.5 to 2.2formed in chains or false heads and often single cell,μm. Macroconidia are falculate, 3 to 5 septate, and 37but there are two cell. Microconidia shape areto 44 4.3 μm. Many chlamydospores are formed aselliptical until pear shape but usually elliptical are oneintermediate and end.side wide and the other side flat and thin.Macroconidia thin, sickle cell is relatively straight,Table 4. Contamination of plants infected with thenarrow and twisted their apical cells and basal cellsfungus isolates of F. proliferatum.are specifically shaped heel. Most of the nidia 1-2 cell: 9( 5-12.5) 2.5(2-3.1) μm.Macroconidia 3 septate: 36 (28-43) 3.5(2.8-4.2),Macroconidia 4 septate: 41(33-45) 3.6(3-4.3) μmand Macroconidia 5 septate: 45(36-50) 3.8(3.4-4.4)IsolateFungiF6F13F26F33Controlrate Infection2.663.334.6630μm. Chlamydospore: Chlamydospores were notTable 5. Contamination of plants infected with theobserved(Fig 1).fungus isolates of F. solaniTable 2. Contamination of plants infected with thefungus isolates of F. proliferatum.Isolate Fungirate te FungiF19F24F5controlrate Infection3.332.662.660Proving pathogenicityIn this study, none of the control pots wilt symptomswere observed, whereas in all the pots treated withfungal strains, there was some degree of wilting. TheTable 3. Contamination of plants infected with thecomparison of all treatments of disease control werefungus isolates of F. solani.significantly different at the one percent level in allIsolateFungiF19F24F5controlrate Infection3.3333.330146 Fatahi et al.treatments, the disease had occurred.Root dip inoculationAfter a week of planting seedlings, irrigated was done.Sampling was started after the appearance of
Int. J. Biosci.2014symptoms.Factors measured: symptoms of chlorosis,mean percentage of infected plants for each isolatereduced growth, wilting plants infected vascular.Thewas calculated and the percentage of infected plantsmean percentage of infected plants for each isolate(symptoms) were determined for each species(Tablewas calculated and the percentage of infected plants4,5).In this experiment, similar results were obtained(symptoms) were determined for each species(Tablewith above experiment.1,2).Pathogens Fusarium proliferatum is more thanDiscussionF. solani.In carnation culture, vascular wilt caused byFusarium is the most important fungal disease(Rattink, 1983). Fusarium crown and root rot incarnation has been reported in USA (Jones et al.,1991). Symptoms were observedon carnationincluded severe vascular browning, root and crownrot. In this study, the species Fusarium proliferatumand Fusarium solani was isolated for the first time onCarnation in Iran. Fusarium was consistently isolatedfrom all wilt affected carnation plants. Garibaldi etal., (2004) and Garibaldi and Minuto (2007), Carveret al., (1996) reported isolation of Fusariumoxysporum f. sp. dianthi and F. avenaceum from wiltFig.chains,1. a,b) Conidiophore with false heads ected greenhouse grown carnation plants inpolyphialide,d)southwest England. Generally the disease incidencesMacroconidiaf)were higher in the high temperature period than inMicroconidia.the low one, and the disease outbreaks were foundonly during the high temperature period in markaziprevious. Nelson et al. (1975) reported the diseaseoccurred especially during the summer when it is ns which favor the disease development.Inoculated carnation plugs developed wilt symptomswithin 30 days after inoculation, symptoms started asyellowing ofleaves, later turned straw coloured,finally wilting and death of the plants was occurred.This is in acceptance with Carver et al., (1996) whoexamined and proved the pathogenicity of Fusariumoxysporum f. sp. dianthi using spore suspensions,introduced into carnation plants via root dip or cutFig. 2. a,b) conidiophore،c) macroconidia،d)microconidia ،e,f) chlamydospores.Soil inoculating spore suspensionAfter a week of planting seedlings, irrigated was done.Sampling was started after the appearance ofsymptoms.Factors measured: symptoms of chlorosis,reduced growth, wilting plants infected vascular.The147 Fatahi et al.stem inoculations. F. proliferatum microconidiameasured Average 9 x 2.5 μm, macroconidia s not observation. The microconidia,macroconidia and chlamydospores of F. onidia Average 40.1 x 4.3 μmand 4.5 x 9.5
Int. J. Biosci.2014μm in diameter. These observations are in accordanceDiseasewith Booth (1971) and Nelson et al., (1983).Minneapolis., USA.ReferenceNelson PE, Pennypacker BW, Toussoun TA,Anonymous.2004. Horticultural Statistics ofRelationships.BurgersHorst RK. 1975. Fusarium stubPublishingdiebackKarnataka State At a Glance, 2002-03. Statisticalcarnation. Phytopathology, 65, e, Govt. of Karnataka, Lalbhag, Bangalore32, 107 p.Ben-YephetNelson PE, Toussoun TA, Marasas WFO. 1983.Y, Reuven M, Genizi A. 1994.Effects of inoculum depth and densityonFusarium species, an iaforUniversityPress,Univ. Park and London. 193.Fusarium wilt in carnations. phytopathology 84,1393-1398.Rattink H. 1983. Spread and control of lt in carnations on artificial substrates 141, 103108.Booth C. 1971. The genus Fusarium. Commonwealthhttp://www.actahort.org/books/141/141 14.htmMycological Institute, Kew Surrey, UK, 142-143 p.Sharma P. 2000. An Integrated approach for theCarver CE, Pitt D, Rhodes DJ. 1996. Aetiologymanagement of Carnation wiltand biological control ofFusarium oxysporum f. sp. dianthi (Pril. And Del.)(DianthusFusarium wilt of pinkscaryophyllus)usingTrichodermacausedbySnyd. And Hans 2001. New Botanist 27, 143-150.aureoviride. Plant Pathology 45, .d01-Sohi HS. 1992 Diseases of ornamental plants in162.xIndia. Publication and information division,IndianCouncil of Agricultural Research, Krishi AnusandhanGaribaldi A, Minuto A, Bertetti D, Gullino ML.Bhavan, New Delhi, India. 46.2004. Fusarium Wilt of Gerbera in Soil and SoillessCrops in Italy. Plant Disease 88, 311.Wheeler BEJ. 1969. An introduction to disease. John Wiley and Sons Ltd., London,UK,pp.301.Garibaldi A, Minuto A. 2007. Fusarium wilt ofgerbera in Spain in soilless crops.PlantDiseaseZhang J, Xingxing W, Yunqing B, Yixin W,91, 638.Guanhui l, Yueqiu H, Zichao M. rt of Fusarium proliferatum Infecting Carnation(Dianthuscaryophyllus L.) in China. JournalJohnson LF, Curl EA, Bond JH, Fribourg HA.of Phytopathology 161, 850-854.1959.Methods for studying soilhttp://dx.doi.org/10.1111/jph.12128148 Fatahi et al.microflora. PlantFirst
Int. J. Biosci. 2014 medium, cream, purple, turning to purple to dark purple ranged. Relatively abundant aerial hyphae and cotton first medium purple and dark purple to white, then changes color. Give spores. After 2 days of culturing the fungus was produces spores on
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