STUDIES ON THE OCCURRENCE OF FUNGI IN A WHEATFIELD . U . FUNGI ON THE PHYLLOPLANE AND FUNGAL AIR-8 PORA

Fungi recorded from northem Ita/y on the phylloplane o{ wheat during its life cycle and the ambient {ungal aiNpora were compared. On the phy/loplane 68 species, be/onging to 35 genera, were identified: Altel'tUII'ia altenfQta, Aureobosidtum pulllllons var. pullulons, Qodosporium cllldos· porioldes and Epicoccum purpurrucens were present al/ months. A few pathogens were occasionally recorded such as Dreschslertz anamorph o{ Coch/iobolus $11/ivus. Erysiphe graminis and &ptorill tritici. In general, a gradual transition in species was noticed from the initial deve/opment to lea{ senescence with an apparent change in {ungal population in ApriL In the air Qodosporium, Alter7UU'Íil, EpicoCCMm and FUSIII'ium were the dominant genera showing high peaks o{ concentration between June and late August. We noticed the greatest abundance o{ {ungi on the phylloplane in April with a relatively low concentration in the air; in June and July the situation was reversed. INTRODUCTION The fint part of this research descnbed the succession of mesoRfúlic. thermophilic and kerati· nophilic micro-fungi in wheatfleld soil (S). This second part compares the fungi on the wheat phylloplane with fungal spores in the surrounding a ir. MATERIALS AND METIIODS Sampling procedures and cultural methods RESUMEN [Estudio .robre la pre.rencia de hongos en campo de trigo. //Hongos del filoplano y esporas fúngica.r aéreas] Se compararon las especies {úngicas detectadas en el filoplano del trigo durante su ciclo de vida con el registro de esporas aéreas ambientales en la zona norte de Italia. Del fi/oplano se aislaron 68 especies pertenecientes a 35 géneros, que inc/u· yeron: Altemaria altemaltl, AUI'eoiHuidium pul/ufans var. pullullms. Oodosporium dodosporioides y EpicoCCMm purpurascens (presentes todos los meses}. Se registraron ocasionalmente unos pocos patógenos tales como Drechslera anamor{o de Cochliobolus SlltiJ1Us, Erysip~ graminis y &ptorio tritici. En general, se aprecia una transición gradual en las especies desde el inicio del desarrollo de la senescencia de la hoja con un aparente cambio de la población fúngica en abril Los géneros dominantes en el aire que mostraron un alto peak de concentración entre junio y agosto co"esponden a Clodosporium, Altemario, Epicoccum y Fusortum. Destacamos la gran abundancia de hongos en abril, con una relatiVa baja concentración en el aire; en junio y julio, la situación fue a la inversa. for isolation of phyUoplane fungi. Wheat leaves were collected monthly from January to July 1983 from plants selected along the diagonal of a wheatfteld plot. A description of the site, chemical data of the soil and climatic data are in the frrst part of this study (5). At each sarnpling 2 leaves from each of 10 wheat seedlings were collected; the leaves were transported within an hour to the laboratory in separate plastic bags, taking care to avoid contarnination, and all mycological processing was carried out on the day of collection. Each leaf was aseptically cut into three segments and subjected to the following three methods: 71 StJtdift Olf tire Oct:III'J'eiU!!I o{ ftuwt"' 11 wlrettt-fiel. n G. Carttta et al. 1) 1eaf impression: the leaf surface of the fust segment was temporarily pressed against malt extract agai" (MEA), arnended with CAF (0.5o/oo) and streptomycin sulphate (1 o/oo), in Petri dishes. The di.shes were incubated at 25° e for 7 days; 2) leaf washing: the second segment of each 1eaf was shaken for 20 min. in 50 cm sterile distilled water. The suspension was diluted at 1/10 and 1/100 and, in duplicate, 0.1 cm of each dilution was spread on Pe tri dishes contai· ninJ MEA. The dishes were incubated at 25 e for 15 days; 3) darnp chamber: each third segment was placed individually in 16 cm diameter plastic dishes containing a 12 cm diameter sterile fllter paper moistened with 5 ml of sterile distilled water containing streptomycin sulphate (40 pg/ml). The dishes were incubated at 25° C for 14-20 days. Al1 resulting fungal colonies were directly identified or transferred to an appropriate medium for identification. Each fungal species was counted only once per dish, even if more than one colony of the same species was present. Sampling of air--spora Spore concentration was determined by a Hirst automatic vo1umetric spore trap (10) installed over the wheat fie1d. lts orifice was at 2 m above ground level. Air was sampled weekly from 1st October 1982 to 30th September 1983. Slides, coated with silicone, were exposed for 24 hours by the trap and then mounted with glycerin jelly containing basic fuchsin as a stain. ·The suction rate of the trap was lcept at 10 1/min throughout the trapping period. The slides moved at 2 mm per hour. We counted the fungal spores along four traverses parallel to the direction of travel at ínter· vals of 500 p in width. The fungal spores were examined by microscopy using 250 magnification. A total of 51 air sampling were made. RESULTS Phylloplane fungi A total of 1557 fungal isolations representa· tive of 68 species belonging to 35 genera were identi· fied. Al1 fungi recorded by the three methods are listed in Table l . Acremoniella atra, GibbereUa acuminata, Gliomastix cerealis, Erysiphe graminis, Septoria tritici 72 and synnematous Hyphomycetes Cephalotrichum stemonitis and Tridturus spiJalis were recorded mainly by the damp chamber method. The leaf washing method encouraged isolation of Aureobasidium pullulans var. puOulans, Bmtanomyces daussenii, Fusarium dimerum, Metschnikowia pul· cherrima, and Torulopsis magnoliae. The fllamentous fungi Botrytis, 1)recbs1era, Epicoccum, Gliocladium, Nigrospora, Tricliodadium, Tridtodenna, Rhizopus and Ulocladium were iso1ated more frequently by the 1eaf impression method. Certain genera were always present, others occurred only occasionally. The genera constantly present were: Alternarla, Aureobasidium, Aspegillus, Cladosporium, Epicoccum and Penicillium. Alternaría, Aspergillus and Oadosporium were each represented by five species with A. alternata, A. fumigatus and C. cladospo· rioides dominant. Penicillium was represented by twelve species with P. aurantio-candidum dominant. The air--spora concentration Results for the air·spora are expressed as monthly averages of spore concentration (Table 2). The mean spore concentration over the period from October 1982 through September 1983 was 4976 spores per cubic metre. Spore concentration showed pronounced seasonal variation. The highest values were found in late spring (June, 14872 spores/m3 ), summer (July, 10293/m3 ) and early autumn. In winter, as in early s~ring, spore concentration was lower (January 519/m and April 798/m3 ). The most common sporetype was Cladosporium, which accounted for 71.39'11. of the total of counted spores. The second most common was Fusarium which amounted to 13.7'11.Altemaria and Epicoccum totalled 2.05"' and 1.97'11. respectively. Al1 other particles recognizable as fungal spores amounted to 10.8% ; most were spores of genera Aspergillus or Penicillium, Botrytis, Drechslera, Ganoderma, Oidium, Polythrincium, Stemphylium, Torula, Ulocladium and Ustilago. Oadosporium spores were regularly :trapped from the wheat field air throughout the year, but mainly from June to August with a peak in June (11797 spo· res/m3 ) . Fungal spores of Fusarium were present all year round but with relatively hlgh incidence in late spring and in summer with a peak in June (2496 spores/m3). Alternarla and Epicoccum showed a similar incidence and concentration: they were found in moderate amounts all year, with the greatest concentrations in October and from July to September, except that Alternarla was absent in January. DISCUSSION In general the fungi of the wheat phylloplane Studie1 on the occurrm« of fll"8i ilt whetzt·field. 11 G. Caretta et al. was composed of ascomycetes and fungi imperfecti. The transitory appearance of sorne genera and species on phylloplane seems signifcant. Botrytis, Cephalotrichum, Oidiodendron, Trichoderma, Septoria, Metschnikowia and Tonalopsis were present until April; Acremonium, Trichocladium, Verticillium did not appear befare April, and Acremoniella, Drechslera, Gliomastix were isolated only in April. Similarly sorne species of the dominant genera occurred until April, e.g. A. Oavus, C. tenuissimum, P. brevi-compactum, P. variabile, S. roseus, Rh. stolonifer, while others were present only frorn April, e.g. A. chlamydospora and C. herbarurn. An important change in the fungal cornposition of leaf surfaces thus seems to occur in April. Flannigan and Campbell (8) in a study of the mycota of the flag leaf, bracts and caryopsis of wheat growing in eastem Scotland found the A. puUulans, C. herbarum, and C. cladosporioides were the earliest established ftlamentous fungi in all organs of wheat; A. altemata and V. lecanii foUowed and E. purpurascens appeared later on the dying leaf and bracts and on the ripening cariopsis. The naturally-occurring yeasts Sporobolomyces and Cryptococcus and the fllamentous fungi Oadosporium, Alternarla, Epiooccum and Aureobasidium are the cornmon leaf saprobes. Their incidence on Gramineae, and their role in controlling plant pathogens, is reported in many investigations (1 , 7, 9). Following the schematic classification of epiphytic fungi on leaves proposed by Dickinson (6), the fungal population on wheat leaves during this investigation consisted mainly of non-pathogenic epiphytes, but sorne species of phytopathological interest were occasionally recorded: Erysiphe graminis, Drechslera anarnorph of Cochliobolus sativus and Septoria tritici. The comparative data between the genera found on wheat phylloplane and the concentration of the fungal air-spora showed sorne discrepancies. The phylloplane appeared to be colonized by different genera very abundant in January, less in February, but increasing in March and April. The fungi decreased with the increasing of the plant growth and in the senescent leaves the number was lower. On the contrary we had the highest spore concentration in the air in June and the lowest in January and March. Oadosporium. Epiooccum and Fusarium behaved in the same rnanner: they showed the greatest abundance on the phylloplane in late winter and in early spring while, at the same time, they had a relatively low concentration in the air. On the other hand these fungi showed the highest concentration in the air in June, July and August when on the phylloplane they were descreasing. Alternarla had the highest occurrence on the leaves in June and the rnax.imum concentration in the air in September. The Fusaria isolated from wheat phylloplane include soil-bome species (F. oxysporum), air-bome species (F. lateritium) and those that Burgess (2) included in the category of Fusaria which are common in soil but have efficient mechanisms for air dispersa! (F. moniliforme). Although F. oxysporum has ·been isolated frorn the air ( 11, 13) it is not weU adapted to air dispersa!. lts presence in the atrnosphere can probably be attributed to wind-blown soil or organic debris (11 , 12) and crop residues stubbles. Dispersa! by rain and water-splash or windblown soil would account for it ability to colonize aerial plant parts. The high concentration of airbome rnacroconidia of Fusarium at Pavia could be correlated with stages in the developrnent of local vegetation and crops. It is interesting to note that sorne of the dominant genera occurring on the wheat leaves Alternarla, Aureobasidium and Epiooccum were rarely isolated from the soil of the same weath field (5). On the other hand very similar pattems on wheat leaves and in wheat soil were observed for Aspergillus, Fusarium and Penicillium. While we could note sorne differences among fungi isolated frorn wheat, maize and rice soils (3, 4, 5) , the fungi recorded from the phylloplane of these three cereals are almost the same. ACKNOWLEDGEMENTS •Research work supported by CNR, Italy, Special grant I.P.R.A. Subproject l. Paper 642. We wish to thank Mrs. G. Delia Volpe Sorrentiní and Mr. L. Morandí for their technical assistance.


INTRODUCTION
The fint part of this research descnbed the succession of mesoRfúlic. thermophilic and kerati· nophilic micro-fungi in wheatfleld soil (S). This second part compares the fungi on the wheat phylloplane with fungal spores in the surrounding a ir. En general, se aprecia una transición gradual en las especies desde el inicio del desarrollo de la senescencia de la hoja con un aparente cambio de la población fúngica en abril Los géneros dominantes en el aire que mostraron un alto peak de concentración entre junio y agosto co"esponden a Clodosporium, Altemario, Epicoccum y Fusortum.

MATERIALS AND METIIODS
Destacamos la gran abundancia de hongos en abril, con una relatiVa baja concentración en el aire; en junio y julio, la situación fue a la inversa.
for isolation of phyUoplane fungi.
Wheat leaves were collected monthly from January to July 1983 from plants selected along the diagonal of a wheatfteld plot. A description of the site, chemical data of the soil and climatic data are in the frrst part of this study (5). At each sarnpling 2 leaves from each of 10 wheat seedlings were collected; the leaves were transported within an hour to the laboratory in separate plastic bags, taking care to avoid contarnination, and all mycological processing was carried out on the day of collection.
Each leaf was aseptically cut into three segments and subjected to the following three methods: 1) 1eaf impression: the leaf surface of the fust segment was temporarily pressed against malt extract agai" (MEA), arnended with CAF (0.5o/oo) and streptomycin sulphate (1 o/oo), in Petri dishes. The di.shes were incubated at 25° e for 7 days; 2) leaf washing: the second segment of each 1eaf was shaken for 20 min. in 50 cm sterile distilled water. The suspension was diluted at 1/10 and 1/100 and, in duplicate, 0.1 cm of each dilution was spread on Pe tri dishes contai· ninJ MEA. The dishes were incubated at 25 e for 15 days; 3) darnp chamber: each third segment was placed individually in 16 cm diameter plastic dishes containing a 12 cm diameter sterile fllter paper moistened with 5 ml of sterile distilled water containing streptomycin sulphate (40 pg/ml). The dishes were incubated at 25° C for 14-20 days.
Al1 resulting fungal colonies were directly identified or transferred to an appropriate medium for identification. Each fungal species was counted only once per dish, even if more than one colony of the same species was present.
Sampling of air--spora Spore concentration was determined by a Hirst automatic vo1umetric spore trap (10) installed over the wheat fie1d. lts orifice was at 2 m above ground level. Air was sampled weekly from 1st October 1982 to 30th September 1983.
Slides, coated with silicone, were exposed for 24 hours by the trap and then mounted with glycerin jelly containing basic fuchsin as a stain. · The suction rate of the trap was lcept at 10 1/min throughout the trapping period. The slides moved at 2 mm per hour. We counted the fungal spores along four traverses parallel to the direction of travel at ínter· vals of 500 p in width. The fungal spores were examined by microscopy using 250 magnification. A total of 51 air sampling were made.
The air--spora concentration Results for the air·spora are expressed as monthly averages of spore concentration ( Table 2). The mean spore concentration over the period from October 1982 through September 1983 was 4976 spores per cubic metre. Spore concentration showed pronounced seasonal variation.
The highest values were found in late spring (June, 14872 spores/m 3 ), summer (July, 10293/m 3 ) and early autumn. In winter, as in early s~ring, spore concentration was lower (January 519/m and April 798/m 3 ). The most common sporetype was Cladosporium, which accounted for 71.39'11. of the total of counted spores. The second most common was Fusarium which amounted to 13.7'11.Altemaria and Epicoccum totalled 2.05"' and 1.97'11. respectively. Al1 other particles recognizable as fungal spores amounted to 10.8% ; most were spores of genera Aspergillus or Penicillium, Botrytis, Drechslera, Ganoderma, Oidium, Polythrincium, Stemphylium, Torula, Ulocladium and Ustilago. Oadosporium spores were regularly :trapped from the wheat field air throughout the year, but mainly from June to August with a peak in June (11797 spo· res/m 3 ) .
Fungal spores of Fusarium were present all year round but with relatively hlgh incidence in late spring and in summer with a peak in June (2496 spores/m3). Alternarla and Epicoccum showed a similar incidence and concentration: they were found in moderate amounts all year, with the greatest concentrations in October and from July to September, except that Alternarla was absent in January.

DISCUSSION
In general the fungi of the wheat phylloplane Studie1 on the occurrm« of fll"8i ilt whetzt·field.  was composed of ascomycetes and fungi imperfecti.
Similarly sorne species of the dominant genera occurred until April, e.g. A. Oavus, C. tenuissimum, P. brevi-compactum, P. variabile, S. roseus, Rh. stolonifer, while others were present only frorn April, e.g. A. chlamydospora and C. herbarurn. An important change in the fungal cornposition of leaf surfaces thus seems to occur in April.
Flannigan and Campbell (8) in a study of the mycota of the flag leaf, bracts and caryopsis of wheat growing in eastem Scotland found the A. puUulans, C. herbarum, and C. cladosporioides were the earliest established ftlamentous fungi in all organs of wheat; A. altemata and V. lecanii foUowed and E. purpurascens appeared later on the dying leaf and bracts and on the ripening cariopsis.
Their incidence on Gramineae, and their role in controlling plant pathogens, is reported in many investigations (1 , 7, 9).
Following the schematic classification of epiphytic fungi on leaves proposed by Dickinson (6), the fungal population on wheat leaves during this investigation consisted mainly of non-pathogenic epiphytes, but sorne species of phytopathological interest were occasionally recorded: Erysiphe graminis, Drechslera anarnorph of Cochliobolus sativus and Septoria tritici.
The comparative data between the genera found on wheat phylloplane and the concentration of the fungal air-spora showed sorne discrepancies. The phylloplane appeared to be colonized by different genera very abundant in January, less in Februa-ry, but increasing in March and April. The fungi decreased with the increasing of the plant growth and in the senescent leaves the number was lower. On the contrary we had the highest spore concentration in the air in June and the lowest in January and March.
Oadosporium. Epiooccum and Fusarium behaved in the same rnanner: they showed the greatest abundance on the phylloplane in late winter and in early spring while, at the same time, they had a relatively low concentration in the air.
On the other hand these fungi showed the highest concentration in the air in June, July and August when on the phylloplane they were descreasing. Alternarla had the highest occurrence on the leaves in June and the rnax.imum concentration in the air in September.
The Fusaria isolated from wheat phylloplane include soil-bome species (F. oxysporum), air-bome species (F. lateritium) and those that Burgess (2) included in the category of Fusaria which are common in soil but have efficient mechanisms for air dispersa! (F. moniliforme). Although F. oxysporum has ·been isolated frorn the air ( 11,13) it is not weU adapted to air dispersa!. lts presence in the atrnosphere can probably be attributed to wind-blown soil or organic debris (11 , 12) and crop residues stubbles. Dispersa! by rain and water-splash or windblown soil would account for it ability to colonize aerial plant parts. The high concentration of airbome rnacroconidia of Fusarium at Pavia could be correlated with stages in the developrnent of local vegetation and crops.
It is interesting to note that sorne of the dominant genera occurring on the wheat leaves Alternarla, Aureobasidium and Epiooccum were rarely isolated from the soil of the same weath field (5). On the other hand very similar pattems on wheat leaves and in wheat soil were observed for Aspergillus, Fusarium and Penicillium.
While we could note sorne differences among fungi isolated frorn wheat, maize and rice so ils (3, 4, 5), the fungi recorded from the phylloplane of these three cereals are almost the same.  Mucor racemosus Fres. 3 . .