Seasonal variation of airborne fungal spore concentrations in a vineyard of North-West Spain. M. Reineria Diaz *, Isabel Iglesias, Victoria Jato. Departamento de ...
Aerobiologia Intermitlomi Journalof Aeroblolo~
ELSEVIER
Aerobiologia 14 (1998) 221-227
Seasonal variation of airborne fungal spore concentrations in a vineyard of North-West Spain M. Reineria Diaz *, Isabel Iglesias, Victoria Jato Departamento de Biologia Vegetaly Cieneia del Suelo, Universidadde Vtgo, Facultad de Ciencias, Campus de Ourense, Edifico Politkcntco, As Lagoas, E-32004 Ourense, Spain
Received 27 November 1996: receivedin revised form 1 April 1998; accepted 8 April 1998
Abstract
Conidial types collected daily in the air above a vineyard in northwest Spain were identified and counted. A total of 26 fungal spore types were recognised: ten of which (Cladosporium, Botrytis, Fusarium-Leptosphaeria type, Torula, Puccinia, Alternaria, Uncinula, Helminthosporium type, Agrocybe and Stemphylium) gave a seasonal total concentration exceeding 1000 spores. Seasonal patterns are shown for 12 of the identified taxa. 9 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Aerobiology; Fungal spectrum; Vineyard; Spain
1. Introduction
Aeromycology, or the study of the biology of airborne fungal propagules, seeks to determine the dispersion, ecology and deposition patterns of phytopathogenic and non-phytopathogenic fungal spores. The capability of producing enormous numbers of spores is characteristic of most moulds (Akers et al., 1979), but they have no well defined seasonal variation models. This makes it difficult to understand their behaviour and works on their geographical distribution are scarce. It is therefore difficult to predict the presence of certain propagules in the environment. Air samplers which capture spores on an inert medium for microscopic identification are generally non-selective, but their identification is seldom possible beyond or even at the genus level. However, they provide important information for the establishment of predictive models. Such studies have not been carried out in Galicia (northwest Spain), so the objective of the present work was to
* Corresponding author. Tel. : + 34 988 387054; fax: + 34 988
387001.
determine which fungal spore types were found with a high incidence over vineyards in our region.
2. Materials and methods
A Lanzoni VPPS 2000 Hirst-type air sampler (flow rate of 10 1/min) was located in a vineyard of the Enology and Viticulture Station at Ponte San ClodioLeiro (Ourense, northwest Spain).The air sampler was located in the center of the vineyard at a height of 1.5 m above ground level and 0.5 m above the maximum height of the vines. Samples were processed as recommended by the Spanish Aerobiology Network (R.E.A.) and collected from 2 June to 29 September 1994, except for a period of 7 days when sampling was prevented by summer storms disrupting the electricity supply. Melinex impregnated with a 2% silicon solution applied using a brush was used as spore-trapping surface. The mounting of the samples was done on glycerol-jelly stained with fucshine. Five longitudinal traverses along the length of the slide were scanned, using the x 100/ 1.30 oil-immersion object lens (Olympus BX50 microscope) to achieve an identification of the fungal spore
0393-5965/98/$ - see front matter 9 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0393-5965(98)00048-1
222
M. Reineria Diaz et al./ Aerobwlogia 14 (1998) 221-227
types as exactly as possible. Using the microscopic field size and flow rate, the daily concentration means were calculated. Ellis (1971), Barnett and Hunter (1972), Ellis (1976), Von Arx (1981), K~/irik et al. (1983)and Grant Smith (1990) were used as reference books for the identification and description of the fungal spore types. The identified spore types were those with morphological characteristics that allowed the most correct microscopical identification Fig. 1. There were other fungal spore types not identified due to important morphological differences shown within the genus that would lead to a high percentage of incorrect identifications. The seasonal patterns of Botrytis, Oidium and Plasmopara spore concentrations were affected by fungicide applications. But this effect will not be discussed, because it is the aim of other studies developed from the same research program.
3. Results
Table 1 shows the predominant identified fungal spore types sampled from the air above the vineyard. The seasonal patterns of eight of the ten spore types which gave total seasonal concentrations exceeding 1000 spores are shown in Fig. 2. Other common types, but with total seasonal concentrations < 1000 spores, are shown in Fig. 3. Spores of the genus Cladosporium Link ex Fr. (Fig. 1A) are from 2-12 to 3-21/lm, ovoid to cylindrical or irregular olivaceous-brown to hyaline and non septate or with one to three septa spores. They are smooth or verrucate, depending on the species and have a basal scar and also 1-3 terminal scars. They may be collected as simple spores or in pairs but are more frequently seen in groups of four or five spores. It was the most abundant fungal spore type collected throughout the period of study reaching, in the middle of August, a maximum daily concentration of 19703 spores/m 3. Botrytis Pers. ex Fr. (Fig. 1B) forms 1-celled smooth, ellipsoidal, ovoid, spherical or subspherical, hyaline to ash-grey conidia, that are 20-25 /~m in length. They were captured throughout the sampling period, but sometimes scarcely exceeding 600 spores/m 3. Largest concentrations occurred on 22 and 23 June 1994, with 1009 and 1004 spores/m 3, respectively. Secondary peaks occurred with values close to 700 spores/m 3. Concentrations tended to decrease over the sampling period with the smallest concentrations in early August and midSeptember. The Fusarium-Leptosphaeria group type included spores of deuteromycetes and ascomycetes with great morphological variability. Fig. 1C shows Leptosphaeria Ces. and de Not.: brownish, multiseptate, slightly
curved spores with one cell larger than the others. This type included spores of Melanomma Nits. ex Fuckel and Phaeosphaeria Miyake with yellow to brown, fusiform spores with traverse septa. Macroconidia with three to five septa, fusiform with thin walls were classified as Fusarium Link ex Fr. They are hyaline and are produced in big numbers. Most species of this group are saprophytic and colonize organic materials and soil, but some are pathogens, mainly affecting Graminaceae. They occur most frequently when vegetation is well developed (Van der Werff, 1967). They appeared constantly throughout the study period, with a daily maximum concentration of 2123 spores/m 3 on 6 June 1994 and smaller secondary peaks during the following months. Torula Pers. ex Fr. (Fig. 1D) has simple or branched chains of dark, spherical-cylindrical cells, sometimes with only two to five brown to olivaceous, smooth or verrucose cells 4-6/~m diameter, Sometimes one of the end cells breaks off to give a characteristically truncated appearance. Concentrations were large at first but then decreased sharply and remained at low concentration. Puccinia Persoon (Fig. 1E) has 1-celled, hyaline to yellowish, cylindrical or ellipsoidal spores, often with spinulose to verrucose ornamentation. The spines arise in the center of an areola or surrounded by a torus, depending on the species. Four to ten equatorial or regularly distributed germ pores may be seen. The spinulose surface may help to disperse these spores in groups (Lacey, 1991). These spores were found in largest concentrations during June, reaching 545 spores/m 3 on 8 June 1994. Afterwards, concentrations decreased but they never disapeared from the air. Alternaria Nees ex Fr. spore type (Fig. 1F) shows great morphological variability. Generally, conidia are muriform, several celled with traverse and longitudinal septa, straight, dark, solitary or in chains of two or few conidia, with smooth to minutely verrucose surface. Alternaria was trapped throughout the period of study but its concentrations varied widely, showing a succession of concentration peaks between the first week of sampling and the middle of August. Oidium Sacc. (Fig. 1G) has ovoid, tip-truncated conidia, 16-29 /~m long. The membrane is colourless and the content is granular with vacuoles. Concentrations were greatest near the start of sampling, but then decreased progressively through the sampling period to < 30 spores/m 3. Helminthosporium type spores (Fig. 1H) included phragmospores of the genera Helminthosporium Link ex Fr., Dreschlera Ito, Bipolaris Shoemaker, Exosporium Link ex Fr. and Sporidesmium Link ex Fr., that were included in the same group because of the difficulty of differentiating them using a light microscope. Conidia are cylindrical with rounded ends or subcylindrical,
M. Reineria Diaz et al./Aerobiologia 14 (1998) 221-227
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Fig. 1. Some of the fungal spore types identified in the area of Ponte San Clodio-Leiro (Ourense-Spain): Cladosporium (1 A), Botrytis (1 B), Leptosphaeria (1 C), Torula (1 D), Puccinia (1 E), Alternaria (1 F), Oidium (1 G), Helminthosporium type (1 H), Stemphylium (1 J), Agrocybe (1 I), Xylariaceae (1 K), Paraphaeosphaeria (1 L), Chaetomium (1 M), Pleospora (1 N), Plasmopara (1 O), Sporormiella (1 P), Helicoma (1 Q), Pithomyces (1 R), Curvularia (1 S), Tilletia (1 T) and Tetraploa (1 U). Magnification 100 x , except (1 U) 40 x ; scale: 10 pm (100 x ) and 10/zm (40 x ). Photographs by R Diaz.
M. Remeria Dia: et al. ,.'Aeroblologla 14 (1998) 221-227
224
curved, straight or ellipsoidal, 50-170/J m long, hyaline to dark brown, sometimes with the cells unequally coloured, the end cells being paler than those intermediate. They show from one to 12 pseudosepta or septa and are mostly smooth, rarely verrucose. They frequently have a prominent, dark brown scar at the base. Helminthosporium type spores were found throughout the study period, The greatest concentration (t80 spores/m 3) was found on 8 August 1994, but there were generally < 100 spores/m 3, with the lowest counts found in late August and early September. Spores of the genus Agrocybe (Fig. lI) are basidiospores with yellowish, solitary spores, 9 • 1210 • 18//m. The walls are smooth and darker than the inner cell in which a darker line may be observed and there is a basal scar. Largest concentrations (145 spores/m 3) were found during the first weeks of sampling in mid June. Afterwards, concentrations were smaller, except for a secondary peak in late August to early September. After this peak, concentrations decreased abruptly and remained very low. Stemphyllium Wallr. (Fig. 1J) is the anamorph of Pleospora Rabehn. (Fig. 1N). Its conidia are pale to mid dark or olivaceous brown with traverse and oblique or longitudinal septa. Their shape is variable but frequently they are ellipsoidal or ovoid and 20-80 /~m long. Some species have one pointed conical apex and the other showing lateral conical protrusions. The walls are smooth, verrucose or echinulate and conidia are often constricted at one or more septa and cicatrized at the base. Concentrations varied during the season, giving two peaks: one, at the beginning (131 spores/m 3 on 21 June 1994) and the other (92 spores/m 3 on 11 August 1994) in the middle of the season. ConTable l Fungal spore types identified in the air of Ponte San Clodio-Leiro (Ourense, Spain) Deuteromycetes
Ascomycetes
Basidiomycetes Oomycetes
Cladosporium Botrytls Fusartum Torula A lternaria
Leptosphaeria Oidium Pleospora Xylariaceae Paraphaeosphaeria Chaetomturn Melanospora
Puccmia Agrocybe Tilletia
Stemphyhum Helminthosporium Arthrimum Helicoma Pithomyces Curvularia Tetraploa Cerebella Zygophiala
Sporormiella
Plasmopara
centrations did not exceed 40 spores/m 3 on other sampling days. There was a further slight increase in the concentration of these spores in the air at the end of the season. Other fnngal spore types that were identified in air samples included those belonging to the family Xylariaceae and the genera Paraphaeosphaeria 0. Eriksson, Pleospora Rabehn., Plasmopara Berl. and de Toni (these shown in Fig. 3), Arthrinium Kunze ex Fr., Sporormiella Elli and Everh. sensu Ahmed and Cain, Helicoma Corda, Pithomyces Ber. and Br. and Curvularia Boedijn, but their concentrations were smaller than those of the ten most abundant spore types (described above), never reaching 1000 spores as the total seasonal concentration. Generally, their concentrations oscillated widely and there were periods in which their presence was not detected in the air. Some spore types never reached a concentration of 10 spores/m 3. Spores of genera Cerebella, Tetraploa Berk. and Br., Melanospora Corda and Zygophyala Mason, appeared sporadically, mainly in later samples.
4. Discussion
In the air of the studied vineyard there is a great variety of airborne fungal spores, some of them directly affecting vines and others acting as pathogens of other cultivate plants. Many of these fungal spores can also affect human health. Spores of the genera Cladosporium, Botrytis, Leptosphaeria, Alternaria and Oidium were all collected in large numbers (K~i~irik et al., 1983). There are many reports of the abundance of Cladosporium spores in the air (Hamilton, 1959; Fern~ndez-Gonz~_lez et al., 1990; Halgawy, 1994). In rural areas, Cladosporium is considered to be the most abundant spore and is related to the proximity to open spaces, particularly to cereal crops (Palmas and Cosentino, 1990). In our work, its concentration was the largest of all the fungal spore types identified in the studied vineyard. The presence of nearby crops also favours the appearance of spores of the genus Torula (Gonzalez et al., 1993), although its concentrations were much smaller than those of Cladosporium spores. Botrytis, Oidium and Plasmopara, which are pathogenic fungal spores, were also found in the study period. Their importance is due to their capability of germinating and provoking fungal diseases in the vines. Although there were fungicide applications in the period of study these spores seemed to appear constantly in the air of the zone, except Plasmopara spores, with the most irregular seasonal pattern of these three vine pathogens. Hamilton (1959) also observed the largest concentrations of Fusarium-Leptosphaeria type spores in early June. The importance of the presence of these spores is
M. Reinerta Dtaz et al. ., Aerobiologta 14 (1998) 2 2 1 - 2 2 7
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Fig. 2. Seasonal concentrations of Cladosporium, Botr),tts, Fusarium-Leptosphama type, Tortda, Puc'cmia, Alternaria, Oidium and Helmmthosporiurn spores m the air of Ponte San Clodio-Letro (Ourense, Spain).
related to their capability of provoking asthma and other respiratory diseases in humans. The spores from genus Torula are 'dry season' spores which arise from mycelia growing in decaying plant materials or from the soil. Finding these concentrations during our period of study may be related to the management activities that were carried out in the
vineyard, producing accumulations of vegetal debris and the existence of neighbouring crops. Spores from Puccinia are also found and are probably related to their pathogenicity to Graminaceae also present in the studied zone. Alternaria spores are typically abundant in the lower levels of the atmosphere, during summer, and may be
226
M. Reineria Diaz et al./Aerobdogia 14 (1998) 221-227
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Fig. 3. Seasonal concentrations of Xylariaceae, Paraphaeosphaerta, Pleospora and Plasmopara spores in the air of Ponte San Clodio-Leiro (Ourense, Spain).
related to the occurrence of plant disease (Simeray et al., 1993). Fern~indez-Gonz~ilez et al. (1990) found a marked seasonal periodicity, with maximum concentrations at the end of spring and in the beginning of summer. We found the most important peaks at the beginning of our monitoring. Helminthosporium type spores reached the lowest concentrations in late August and early September, and Stemphylium concentrations, after having reached their maximum during summer, should remain at a lower level for the remainder of the year (Gonz~ilez et al., 1993). These results for Helminthosporium type spores are different from those reported by Hamilton (1959) for the same genus. The appareance of the other identified spore types is scarce. Their concentrations may be better expressed as presence/absence due to their maximum values of 1 or 2 spores/m 3 during the period of study. Their importance in the air of the zone is much less important than that of the previous spore types, but the aim of this study was to identify as much spore types as possible in our region. They were therefore identifed and taken into account.
Acknowledgements The authors would like to thank the Xunta de
Galicia for the XUGA-38302B95 project and to the Estacidn de Enoloxia e Viticultura at Ponte San Clodio-Leiro (Ourense) for its valuable collaboration.
References Akers TG, Edmonds RL, Kramer C L et al. Sources and characteristics of airborne materials. In: Edmonds RL, editor. Aerobiology: the ecological systems approach. Sroundsburg, PA: Dowden, Hutchinson, Ross, 1979. Barnett HL, Hunter BB. Illustrated Genera of Imperfect Fungi. 3rd ed. Burgess, MN, USA, 1972. Ellis MB. Dematiaceous Hyphomycetes. UK: Commonwealth Mycological Institute, 1971. Ellis MB. More Dematiaceous Hyphomycetes. UK: Commonwealth Mycological Institute, 1976. Fern~indez-Gonz~tlez D, Su~irez-Cervera M, Diaz-Gonz~lez TE, Valencia-Barrera RM. Airborne pollen and spores of Le6n (Spain). Abstracts 1990. Gonz~ilez F J, Gonz~ilez ML, Romero F, Pineda F, Candau P. Airborne fungal spores trend over a highly polluted area of southwest Spain using Cour's trap. Aerobiologia 1993;9:39 45. Grant Smith E. Sampling and identifying allergenic pollen and molds. San Anton]o, TX: Blewstone Press, 1990. Halgawy MH. Fungal airspora of Kuwait city, Kuwait, 19751987. Grana 1994;33:340-5. Hamilton ED. Studies on the air spora. Acta Allergol 1959;13:157-71.
M. Reinerla Diaz et al./Aerobiologia 14 (1998) 221-227
K~/irik A, Keller J, Kiffer E, Perreau J, Reisinger O. Atlas of airborne fungal spores in Europe. In: Nilsson S, editor. Springer-Verlag: Berlin, 1983. Lacey J. Aggregation of spores and its effect on aerodynamic behaviour. Grana 1991;30:438-9. Palmas F, Cosentino S. Comparison between fungaI airspore concentration at two different sites in the south of Sardinia. Grana 1990;29:88-93.
227
Simeray J, Chaumont JP, L~ger D. Seasonal variations in the airborne fungal spore population on the East of France (Franche-Comtr): comparison between urban and rural environment during 2 years. Aerobiologia 1993;9:201-5. Van der Werff PJ. Daily census of fungus spores. Rev Palaeobotan PalynoI t967:4:203-26. Von Arx JA. In: Cramer J, editor. The Genera of Fungi Spornlating in Pure Culture, 3, 1981.
