Fungal isolations were made from leaves of tomato plants cultivated in greenhouses in an area close to La Plata, Argentina. Three different schemes of fungicide application were evaluated: high frequency preventive sprayings (Commercial Greenhouse I), low frequency preventive applications (Commercial Greenhouse II) and no fungicide spraying (Control Greenhouse). Leaves were sampled immediately after second fruit formation from three levels of the foliage: low, medium and high. Plating dilution was used to isolate fungal species. Total c.f.u. number and species composition and diversity were assessed by the plating dilution technique. Fungal populations were most abundant on leaves from lower parts of the foliage in the Control Greenhouse. Diversity varied according to fungicide application frequency and leaf position in the canopy. Higher values were recorded for lower leaves in the Control Greenhouse compared with upper leaves from Commercial Greenhouse II. Likewise position in the canopy influenced the frequency of some species. The implications for natural biological control are discussed.Centro de Investigaciones de Fitopatologí

Alippi, Héctor E.

Mittidieri, I.

Mónaco, Cecilia Inés

Nico, A. I.

Acta Agronomica Hungarica

SEDICI - Repositorio de la UNLP

Acta Agronomica Hungarica, 49(3), pp. 243–250 (2001)
0238–0161/2001/$ 5.00©2001 Akadémiai Kiadó, Budapest
SAPROPHYTIC FUNGI ON TOMATO PHYLLOPLANE:
EFFECT OF FUNGICIDES AND LEAF POSITION ON
ABUNDANCE, COMPOSITION AND DIVERSITY
C. I. MÓNACO, 1,3 A. I. NICO,1 H. ALIPPI1 and I. MITTIDIERI 2
1CENTRO DE INVESTIGACIONES DE FITOPATOLOGÍA (CIDEFI). FACULTAD DE CIENCIAS
AGRARIAS Y FORESTALES, UNIVERSIDAD NACIONAL DE LA PLATA, LA PLATA, ARGENTINA
2ESTACIÓN EXPERIMENTAL SAN PEDRO, INSTITUTO NACIONAL DE TECNOLOGÍA
AGROPECUARIA, ARGENTINA
3 COMISIÓN DE INVESTIGACIONES CIENTÍFICAS DE LA PROVINCIA DE BUENOS AIRES,
ARGENTINA
Received: 25 January, 2001; accepted: 18 July, 2001
Fungal isolations were made from leaves of tomato plants cultivated in greenhouses
in an area close to La Plata, Argentina. Three different schemes of fungicide application
were evaluated: high frequency preventive sprayings (Commercial Greenhouse I), low
frequency preventive applications (Commercial Greenhouse II) and no fungicide spraying
(Control Greenhouse). Leaves were sampled immediately after second fruit formation
from three levels of the foliage: low, medium and high. Plating dilution was used to isolate
fungal species. Total c.f.u. number and species composition and diversity were assessed by
the plating dilution technique. Fungal populations were most abundant on leaves from
lower parts of the foliage in the Control Greenhouse. Diversity varied according to
fungicide application frequency and leaf position in the canopy. Higher values were
recorded for lower leaves in the Control Greenhouse compared with upper leaves from
Commercial Greenhouse II. Likewise position in the canopy influenced the frequency of
some species. The implications for natural biological control are discussed.
Key words: biodiversity, biological control, phylloplane, tomato
Introduction
Tomato is one of the most important vegetable crops cultivated in the area
of La Plata, Argentina (35º W, 58º S). In commercial greenhouses growers
usually develop high input cropping systems in which foliar fungal diseases are
controlled by high frequency spraying with preventive fungicides. It has been
suggested that the excessive use of fungicides may result in a human health risk
both for consumers and for farmers living in the area (De Waard et al., 1993).
According to Bollen (1971), fungicide application has two main drawbacks: the
introduction of large quantities of toxic substances into ecosystems and the
generation of fungicide-resistant strains.
Biological control has been suggested as a suitable method for decreasing
the incidence and severity of several foliar diseases (Mónaco et al., 1999). The
use of biological control in integrated disease management programmes might
result in a decrease in fungicide applications, thus leading to a reduction in
pollution.
C. I. MÓNACO et al.
Acta Agronomica Hungarica, 49, 2001
244
Regarding biological control in the phylloplane most research has
addressed the introduction of foreign antagonistic microorganisms (Blakeman
and Fokkema, 1982). Other authors have outlined the importance of indigenous
antagonists in suppressing disease and the managing of ecosystem conditions, in
order to enhance biological control (Marois and Rouse, 1991). Saprobic fungi
inhabiting the phylloplane may exert antagonistic effects on pathogenic fungi, by
competition, antibiosis and/or parasitism (Marois and Coleman, 1994).
Therefore, microscopic fungi may play an important role not only in
pathogenesis but also in the control of foliar diseases (Fokkema, 1993; Andrews
and Kenerly, 1978). Restriction or delay in disease onset may be due to the
activity of antagonists, which is why the saprophytic fungi of the phylloplane are
of interest for natural biological control.
The ability of the indigenous microbial population to prevent pathogenic
infections depends mainly on its abundance and diversity. Diversity is important
for the effective colonization of different microniches in ecosystems where
pathogenesis might take place (Baker, 1991).
Many events that take place on leaf surfaces affect the native saprophytic
fungi, quantitatively and qualitatively. A knowledge of the factors that affect the
diversity and growth of saprophytic fungi on leaf surfaces would allow us to
design a cropping system that would enhance the abundance and diversity of
natural antagonists.
The purpose of the work reported here was to evaluate and identify
species of fungi inhabiting the phylloplane of tomato from greenhouses with
different kinds of management (high input, low input and no pesticide
application) and to assess the effect of fungicide application schemes on the
abundance and diversity of saprophytic fungi at different canopy levels.
Materials and methods
Sampling of phylloplane fungal flora
Samples were collected from tomato plants cultivated in greenhouses at three different
locations in the La Plata green belt. The first sampling was made in a highly technified farm, with
five commercial greenhouses devoted to tomato and pepper, managed under a large-scale
commercial scheme (Commercial Greenhouse I). The second sampling location was at a smaller
farm managed by a family with two greenhouses for tomato cropping (Commercial Greenhouse
II). The fungicide application schemes in the two locations are detailed in Figure 1. The third
sampling was made in the experimental greenhouse of the Facultad de Ciencias Agrarias y
Forestales in La Plata with no pesticide application. Samples were collected in five places
equidistantly located along an oblique line with respect to the rows. One plant was selected at each
sampling site and three leaves were detached from each: a lower leaf from the third node, a middle
leaf from the sixth node and an upper leaf from the ninth node. The combination of fungicide
application schemes and leaf canopy positions resulted in nine different treatments.
SAPROPHYTIC FUNGI ON TOMATO PHYLLOPLANE
Acta Agronomica Hungarica, 49, 2001
245
  Sept. 1st             Oct. 1st       Sampling date   Nov. 1st
Mz Cl Cb Mz Cl
Cb Cl Cb Cl
  Sept. 1st             Oct. 1st          Sampling date    Nov. 1s
Mz Mz
Commercial Greenhouse I
Commercial Greenhouse II
Cb
Fig. 1. Fungicide application schemes in the two Commercial Greenhouses. [Cb: Carbendazim
(BENCARB®, WP 50%); Mz: Mancozeb (DITHANE® WP 80%); Cl: Chlorotalonil (DACONIL® F 72%)]
Isolation of fungal species
Each leaf sample was dissected into four folioles (subsamples). Then five disks 2 cm2 in
size (16 mm in diameter) were cut from each subsample. These five disks (10 cm2) were put in 100
ml of sterile distilled water and the mixture was shaken in a magnetic shaker at 120 rev. min-1 for
30 min. The suspension was diluted tenfold in sterile distilled water. One ml of this dilution was
poured into a Petri dish (9 cm in diameter) to which 10 ml of 2% PDA, oxgall (4 g/l) and 1 ml of
streptomycin sulphate solution (250 mg/l) were added. The dishes were incubated at 25±2ºC in the
dark for 7 days, after which the number of colony forming units (c.f.u.) was assessed and the
species were identified.
The experiments were repeated at least twice with at least four replicates. As the results
were the same among experiments, statistical analyses were applied to the last experiments.
The data were statistically analysed by ANOVA and the Tukey multiple range test
(P<0.05).
Diversity
In order to assess the biodiversity of the fungal populations the Shannon and Weaver
diversity index (Odum, 1985) was calculated based on the replicates of each treatment. The results
were statistically analysed as described above.
C. I. MÓNACO et al.
Acta Agronomica Hungarica, 49, 2001
246
Results
The highest number of fungi was found on the lowest leaves of plants
grown in greenhouses that had not been treated (Control Greenhouse) (Figure 2).
The other treatments resulted in lower levels of fungal populations, except for
leaves located in the upper part of the canopy from Commercial Greenhouse I.
Leaves from the medium and lower strata of the canopy from the Control
Greenhouse showed the highest diversity indexes, while upper leaves from
Commercial Greenhouse II had the least diverse fungal populations (Figure 3).
Fig. 2. Abundance of fungal populations on tomato phylloplane. Columns with the same letter do
               not differ significantly on the basis of Tukey´s multiple range test (P<0.05)
Fig. 3. Diversity of fungal populations (Shannon & Weber index) on tomato phylloplane. Columns
with the same letter do not differ significantly on the basis of  Tukey´s multiple range test (P<0.05)
ab
abc
a
ab
a
ab
c
ab ab
0
10
20
30
40
50
60
70
80
90
100
Control Comm. I Comm. II
Fu
ng
al
 a
bu
nd
an
ce
 (c
.f.
u.
/le
af
 c
m
2 )
Upper
Medium
Lower
c
b
a
d
b
c
d
a
c
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
3 .0
3 .5
4 .0
C o n tro l C o m m . I C o m m . II
Di
ve
rs
ity
 in
de
x
U p p er
M e d iu m
L o w er
SAPROPHYTIC FUNGI ON TOMATO PHYLLOPLANE
Acta Agronomica Hungarica, 49, 2001
247
Most of the isolated species were more abundant on tomato leaves from
the Control Greenhouse and Commercial Greenhouse II (Table 1). Cryptococcus
luteolus, Rhodotorula sp., Arthrinium phaeospermum, Aspergillus sp (1),
Trichoderma harzianum (1), T. polysporum, Penicillium sp. (1), Alternaria
alternata and Cladosporium cladosporioides had a significantly greater c.f.u.
number in the Control Greenhouse than in the other locations. However, the
c.f.u. number for some Penicillium and Aspergillus species was greater in
Commercial Greenhouse I.
Leaf position in the canopy influenced the fungal population structure.
Some species, such as Epicoccum nigrum, Chaetomium globosum, Aspergillus
sp. (1), Trichoderma harzianum (1), T. polysporum and Penicillium spp., were
more abundant on lower leaves, while Alternaria alternata, Cryptococcus
luteolus, Rhodotorula sp., Pleospora herbarum, Fusarium semitectum, F.
oxysporum and Cladosporium cladosporioides were present in greater numbers
on leaves located in the upper and medium levels.
Table 1
Frequency of saprophytic fungus species on the phylloplane
c.f.u./leaf cm2 (1)
Species Upper leaves Medium leaves Lower leaves
CG I CG II Control CG I CG II Control CG I CG II Control
Alternaria alternata 0 0 10 0 1.1 2.6 2.6 0 3.6 *
Arthrinium phaeospermum 0 0 0 0 1.6 1.8 0 2.4 3.2 *
Aspergillus niger 0 0 2.6 0 2.3 3.0 * 0 3.6 * 2.7
Aspergillus sp. (1) 3.7 1.1 0 1.4 1.5 1.4 1.2 2.8 2.7
Aspergillus sp. (2) 0 0 0 0 0 0 0 0 3.6 *
Cladosporium cladosporioides 1.5 5.04 5.6 0 3.0 4.7 1.3 4.6 6.4
Cryptococcus luteolus 0 0 10.6 0 2.6 9.5 * 0 1.04 5.4 *
Chaetomium globosum 0 0 0 0 0 3.6 0 1.24 5.6 *
Epicoccum nigrum 0 0 1 0 1.2 1.9 2.4 0 3.6 *
Fusarium oxysporum 0 0 3.7 0 0.98 1.9 0 0 1.16
F. semitectum 0 6.2 0.4 1.4 0 1.9 0 0 2.7 *
Nigrospora sp. 1.4 0 0 0 0.8 1.4 0 1.6 2.7 *
Penicillium sp. (1) 28 13.25 32.6 10.2 12.3 10.4 29 13.7 20.4 *
Penicillium sp. (2) 19 12 4.6 19.1 * 9.8 19.1 * 20.4 * 14.8 17.06
Rhodotorula sp. 0 0 3.7 0 0.8 5.04 * 0 0.5 3.0
Pleospora herbarum 0 0 2.9 0 0 3.6 * 0 0 0
Trichoderma harzianum (1) 0 0 0 0 0 2.1 0 0.8 5.6 *
T. harzianum (2) 0 0 0 0 0 1 0 0 3.1 *
T. polysporum 2.6 0 2.8 5 0 3.6 0 5.8 10.2 *
(1) Values expressed in thousands; CG I: Commercial Greenhouse I, CG II: Commercial Greenhouse
II.; *Values significantly greater than others in the same row according to the Tukey test (P<0.05).
C. I. MÓNACO et al.
Acta Agronomica Hungarica, 49, 2001
248
Discussion
Both population diversity and fungal abundance were affected by leaf
position in the canopy. Fluctuations in temperature and relative humidity
decrease towards lower positions in the canopy (Blakeman, 1985). Andrews
(1991) suggested the importance of UV light intensity in the regulation of fungal
populations in the canopy. This might explain the lower number of fungi
recorded on leaves in the upper part of the canopy in both the Control
Greenhouse and Commercial Greenhouse II. According to Odum (1985)
biodiversity is low in physically controlled ecosystems, i.e. in those subjected to
strongly limited physical factors. Leaves located within the canopy might
provide a less restricted environment, thus resulting in more diverse
communities. Based on the Control Greenhouse and Commercial Greenhouse II,
it was concluded that the fungal population was more abundant on lower leaves,
while medium and upper leaves had similar fungal populations. According to
Andrews (1991) the presence of abundant nutrient sources in lower leaves due to
the proximity of the soil might also explain the presence of higher numbers of
fungi. On the other hand, Ruinen (1961) asserted that the successional climax of
phylloplane microbial populations could not be reached until the leaves reached
maturity, and the lower leaves are the most mature ones on the plant. These
findings regarding abundance and diversity and their association with position
within the canopy, confirm the results obtained for the Control Greenhouse and
Commercial Greenhouse II, but not those found for Commercial Greenhouse I,
where the upper leaves had more developed fungal populations than any of the
other sprayed leaves. The longer time which elapsed (30 days) from the last
application in Commercial Greenhouse I might explain this phenomenon. It is
important to consider both abundance and diversity when planning natural
biological control (Cook and Baker, 1989). So the abundance and diversity of
saprophytic fungi achieved in the Control Greenhouse might be more suitable
for appropriate disease suppression.
The qualitative composition of the fungal population was also affected by
the fungicide application scheme and leaf position in the canopy. Most of the
yeasts and fungi found in the upper leaves belong to the naturally-occurring
resident microorganisms on aerial plant surfaces listed by Blakeman and
Fokkema (1982). The phyllosphere of the lower leaves is inhabited by a number
of soil fungi, mainly Trichoderma spp. and Chaetomium globosum. The
ubiquitous genera Aspergillus and Penicillium have a distribution profile
unrelated with leaf position in the canopy. The effect of the fungicide application
scheme on the saprophytic fungus composition allows a distinction to be made
between fungicide-tolerant species, i.e. those that prevailed and persisted on the
leaves from the commercial greenhouses, and susceptible species, which were
present only in the Control Greenhouse. The results demonstrated that fungicide
application led to the suppression of many potential antagonists of foliar
SAPROPHYTIC FUNGI ON TOMATO PHYLLOPLANE
Acta Agronomica Hungarica, 49, 2001
249
pathogens. Resident yeasts, described by many authors as effective biocontrol
agents in the phylloplane (Suzzi et al., 1995; Fokkema and Van der Meulen,
1976) were the most affected by fungicide application. The same was valid for
Trichoderma spp., an acknowledged biocontrol agent whose population was
particularly affected by fungicide applications.
The prevalence of Aspergillus spp. and Penicillium spp. on leaves from
Commercial Greenhouses I and II suggests that fungicide resistance may be the
result of the selection pressure performed on both genera (Bollen, 1971). The
capacity of some indigenous saprobic phylloplane fungi to develop resistance to
fungicides is a desirable characteristic which could be utilised in biocontrol
methods (Fokkema and Naaj, 1981; Fokkema, 1993).
Acknowledgements
The authors wish to thank Dr. Pedro Balatti and Mr. M. R. Simón for their critical reading
of the manuscript.
References
Andrews, J., Kennerly, C. (1978): The effect of a pesticide program on non target epiphytic
microbial populations of apple leaves. Canadian Journal of Microbiology, 24, 1058–1072.
Andrews, J. (1991): Biological control in the phyllosphere. Annual Review of Phytopathology, 30,
603–635.
Baker, R. (1991): Diversity in biological control. Crop Protection, 10, 85–94.
Blakeman, J., Fokkema, N. (1982): Potential for biological control of plant diseases on the
phylloplane. Annual Review of Phytopathology, 20, 167–192
Blakeman, J. (1985): Ecological succession of leaf surface microorganisms in relation to
biological control. pp. 63–82. In: Windels, C., Lindow, S. (eds.), Biological Control in the
Phylloplane. APS Press, Saint Paul, USA.
Bollen, G. (1971): Resistance to benomyl and some chemically related compounds in strains of
Penicillium species. Netherland Journal of Plant Pathology, 77, 187–193.
Cook, R., Baker, K. (1989): The Nature and Practice of Biological Control of Plant Pathogens.
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(1993): Chemical control of plant diseases: Problems and prospects. Annual Review of
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Fokkema, N. (1993): Opportunities and problems of control of foliar pathogens with
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Morris, C., Rouse, D. (1991): Role of nutrients in regulating epiphytic bacterial populations. pp.
63–82. In: Windels, C., Lindow, S. (eds.), Biological Control in the Phylloplane. APS
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English

Saprophytic fungi on tomato phylloplane: Effect of fungicides and leaf position on abundance, composition and diversity

Servicio de Difusión de la Creación Intelectual

Acta Agronomica Hungarica, 49(3), pp. 243–250 (2001)0238–0161/2001/$ 5.00©2001 Akadémiai Kiadó, BudapestSAPROPHYTIC FUNGI ON TOMATO PHYLLOPLANE:EFFECT OF FUNGICIDES AND LEAF POSITION ONABUNDANCE, COMPOSITION AND DIVERSITYC. I. MÓNACO, 1,3 A. I. NICO,1 H. ALIPPI1 and I. MITTIDIERI 21CENTRO DE INVESTIGACIONES DE FITOPATOLOGÍA (CIDEFI). FACULTAD DE CIENCIASAGRARIAS Y FORESTALES, UNIVERSIDAD NACIONAL DE LA PLATA, LA PLATA, ARGENTINA2ESTACIÓN EXPERIMENTAL SAN PEDRO, INSTITUTO NACIONAL DE TECNOLOGÍAAGROPECUARIA, ARGENTINA3 COMISIÓN DE INVESTIGACIONES CIENTÍFICAS DE LA PROVINCIA DE BUENOS AIRES,ARGENTINAReceived: 25 January, 2001; accepted: 18 July, 2001Fungal isolations were made from leaves of tomato plants cultivated in greenhousesin an area close to La Plata, Argentina. Three different schemes of fungicide applicationwere evaluated: high frequency preventive sprayings (Commercial Greenhouse I), lowfrequency preventive applications (Commercial Greenhouse II) and no fungicide spraying(Control Greenhouse). Leaves were sampled immediately after second fruit formationfrom three levels of the foliage: low, medium and high. Plating dilution was used to isolatefungal species. Total c.f.u. number and species composition and diversity were assessed bythe plating dilution technique. Fungal populations were most abundant on leaves fromlower parts of the foliage in the Control Greenhouse. Diversity varied according tofungicide application frequency and leaf position in the canopy. Higher values wererecorded for lower leaves in the Control Greenhouse compared with upper leaves fromCommercial Greenhouse II. Likewise position in the canopy influenced the frequency ofsome species. The implications for natural biological control are discussed.Key words: biodiversity, biological control, phylloplane, tomatoIntroductionTomato is one of the most important vegetable crops cultivated in the areaof La Plata, Argentina (35º W, 58º S). In commercial greenhouses growersusually develop high input cropping systems in which foliar fungal diseases arecontrolled by high frequency spraying with preventive fungicides. It has beensuggested that the excessive use of fungicides may result in a human health riskboth for consumers and for farmers living in the area (De Waard et al., 1993).According to Bollen (1971), fungicide application has two main drawbacks: theintroduction of large quantities of toxic substances into ecosystems and thegeneration of fungicide-resistant strains.Biological control has been suggested as a suitable method for decreasingthe incidence and severity of several foliar diseases (Mónaco et al., 1999). Theuse of biological control in integrated disease management programmes mightresult in a decrease in fungicide applications, thus leading to a reduction inpollution.C. I. MÓNACO et al.Acta Agronomica Hungarica, 49, 2001244Regarding biological control in the phylloplane most research hasaddressed the introduction of foreign antagonistic microorganisms (Blakemanand Fokkema, 1982). Other authors have outlined the importance of indigenousantagonists in suppressing disease and the managing of ecosystem conditions, inorder to enhance biological control (Marois and Rouse, 1991). Saprobic fungiinhabiting the phylloplane may exert antagonistic effects on pathogenic fungi, bycompetition, antibiosis and/or parasitism (Marois and Coleman, 1994).Therefore, microscopic fungi may play an important role not only inpathogenesis but also in the control of foliar diseases (Fokkema, 1993; Andrewsand Kenerly, 1978). Restriction or delay in disease onset may be due to theactivity of antagonists, which is why the saprophytic fungi of the phylloplane areof interest for natural biological control.The ability of the indigenous microbial population to prevent pathogenicinfections depends mainly on its abundance and diversity. Diversity is importantfor the effective colonization of different microniches in ecosystems wherepathogenesis might take place (Baker, 1991).Many events that take place on leaf surfaces affect the native saprophyticfungi, quantitatively and qualitatively. A knowledge of the factors that affect thediversity and growth of saprophytic fungi on leaf surfaces would allow us todesign a cropping system that would enhance the abundance and diversity ofnatural antagonists.The purpose of the work reported here was to evaluate and identifyspecies of fungi inhabiting the phylloplane of tomato from greenhouses withdifferent kinds of management (high input, low input and no pesticideapplication) and to assess the effect of fungicide application schemes on theabundance and diversity of saprophytic fungi at different canopy levels.Materials and methodsSampling of phylloplane fungal floraSamples were collected from tomato plants cultivated in greenhouses at three differentlocations in the La Plata green belt. The first sampling was made in a highly technified farm, withfive commercial greenhouses devoted to tomato and pepper, managed under a large-scalecommercial scheme (Commercial Greenhouse I). The second sampling location was at a smallerfarm managed by a family with two greenhouses for tomato cropping (Commercial GreenhouseII). The fungicide application schemes in the two locations are detailed in Figure 1. The thirdsampling was made in the experimental greenhouse of the Facultad de Ciencias Agrarias yForestales in La Plata with no pesticide application. Samples were collected in five placesequidistantly located along an oblique line with respect to the rows. One plant was selected at eachsampling site and three leaves were detached from each: a lower leaf from the third node, a middleleaf from the sixth node and an upper leaf from the ninth node. The combination of fungicideapplication schemes and leaf canopy positions resulted in nine different treatments.SAPROPHYTIC FUNGI ON TOMATO PHYLLOPLANEActa Agronomica Hungarica, 49, 2001245  Sept. 1st             Oct. 1st       Sampling date   Nov. 1stMz Cl Cb Mz ClCb Cl Cb Cl  Sept. 1st             Oct. 1st          Sampling date    Nov. 1sMz MzCommercial Greenhouse ICommercial Greenhouse IICbFig. 1. Fungicide application schemes in the two Commercial Greenhouses. [Cb: Carbendazim(BENCARB®, WP 50%); Mz: Mancozeb (DITHANE® WP 80%); Cl: Chlorotalonil (DACONIL® F 72%)]Isolation of fungal speciesEach leaf sample was dissected into four folioles (subsamples). Then five disks 2 cm2 insize (16 mm in diameter) were cut from each subsample. These five disks (10 cm2) were put in 100ml of sterile distilled water and the mixture was shaken in a magnetic shaker at 120 rev. min-1 for30 min. The suspension was diluted tenfold in sterile distilled water. One ml of this dilution waspoured into a Petri dish (9 cm in diameter) to which 10 ml of 2% PDA, oxgall (4 g/l) and 1 ml ofstreptomycin sulphate solution (250 mg/l) were added. The dishes were incubated at 25±2ºC in thedark for 7 days, after which the number of colony forming units (c.f.u.) was assessed and thespecies were identified.The experiments were repeated at least twice with at least four replicates. As the resultswere the same among experiments, statistical analyses were applied to the last experiments.The data were statistically analysed by ANOVA and the Tukey multiple range test(P<0.05).DiversityIn order to assess the biodiversity of the fungal populations the Shannon and Weaverdiversity index (Odum, 1985) was calculated based on the replicates of each treatment. The resultswere statistically analysed as described above.C. I. MÓNACO et al.Acta Agronomica Hungarica, 49, 2001246ResultsThe highest number of fungi was found on the lowest leaves of plantsgrown in greenhouses that had not been treated (Control Greenhouse) (Figure 2).The other treatments resulted in lower levels of fungal populations, except forleaves located in the upper part of the canopy from Commercial Greenhouse I.Leaves from the medium and lower strata of the canopy from the ControlGreenhouse showed the highest diversity indexes, while upper leaves fromCommercial Greenhouse II had the least diverse fungal populations (Figure 3).Fig. 2. Abundance of fungal populations on tomato phylloplane. Columns with the same letter do               not differ significantly on the basis of Tukey´s multiple range test (P<0.05)Fig. 3. Diversity of fungal populations (Shannon & Weber index) on tomato phylloplane. Columnswith the same letter do not differ significantly on the basis of  Tukey´s multiple range test (P<0.05)ababcaabaabcab ab0102030405060708090100Control Comm. I Comm. IIFungal abundance (c.f.u./leaf cm2 )UpperMediumLowercbadbcdac0 .00 .51 .01 .52 .02 .53 .03 .54 .0C o n tro l C o m m . I C o m m . IIDiversity indexU p p erM e d iu mL o w erSAPROPHYTIC FUNGI ON TOMATO PHYLLOPLANEActa Agronomica Hungarica, 49, 2001247Most of the isolated species were more abundant on tomato leaves fromthe Control Greenhouse and Commercial Greenhouse II (Table 1). Cryptococcusluteolus, Rhodotorula sp., Arthrinium phaeospermum, Aspergillus sp (1),Trichoderma harzianum (1), T. polysporum, Penicillium sp. (1), Alternariaalternata and Cladosporium cladosporioides had a significantly greater c.f.u.number in the Control Greenhouse than in the other locations. However, thec.f.u. number for some Penicillium and Aspergillus species was greater inCommercial Greenhouse I.Leaf position in the canopy influenced the fungal population structure.Some species, such as Epicoccum nigrum, Chaetomium globosum, Aspergillussp. (1), Trichoderma harzianum (1), T. polysporum and Penicillium spp., weremore abundant on lower leaves, while Alternaria alternata, Cryptococcusluteolus, Rhodotorula sp., Pleospora herbarum, Fusarium semitectum, F.oxysporum and Cladosporium cladosporioides were present in greater numberson leaves located in the upper and medium levels.Table 1Frequency of saprophytic fungus species on the phylloplanec.f.u./leaf cm2 (1)Species Upper leaves Medium leaves Lower leavesCG I CG II Control CG I CG II Control CG I CG II ControlAlternaria alternata 0 0 10 0 1.1 2.6 2.6 0 3.6 *Arthrinium phaeospermum 0 0 0 0 1.6 1.8 0 2.4 3.2 *Aspergillus niger 0 0 2.6 0 2.3 3.0 * 0 3.6 * 2.7Aspergillus sp. (1) 3.7 1.1 0 1.4 1.5 1.4 1.2 2.8 2.7Aspergillus sp. (2) 0 0 0 0 0 0 0 0 3.6 *Cladosporium cladosporioides 1.5 5.04 5.6 0 3.0 4.7 1.3 4.6 6.4Cryptococcus luteolus 0 0 10.6 0 2.6 9.5 * 0 1.04 5.4 *Chaetomium globosum 0 0 0 0 0 3.6 0 1.24 5.6 *Epicoccum nigrum 0 0 1 0 1.2 1.9 2.4 0 3.6 *Fusarium oxysporum 0 0 3.7 0 0.98 1.9 0 0 1.16F. semitectum 0 6.2 0.4 1.4 0 1.9 0 0 2.7 *Nigrospora sp. 1.4 0 0 0 0.8 1.4 0 1.6 2.7 *Penicillium sp. (1) 28 13.25 32.6 10.2 12.3 10.4 29 13.7 20.4 *Penicillium sp. (2) 19 12 4.6 19.1 * 9.8 19.1 * 20.4 * 14.8 17.06Rhodotorula sp. 0 0 3.7 0 0.8 5.04 * 0 0.5 3.0Pleospora herbarum 0 0 2.9 0 0 3.6 * 0 0 0Trichoderma harzianum (1) 0 0 0 0 0 2.1 0 0.8 5.6 *T. harzianum (2) 0 0 0 0 0 1 0 0 3.1 *T. polysporum 2.6 0 2.8 5 0 3.6 0 5.8 10.2 *(1) Values expressed in thousands; CG I: Commercial Greenhouse I, CG II: Commercial GreenhouseII.; *Values significantly greater than others in the same row according to the Tukey test (P<0.05).C. I. MÓNACO et al.Acta Agronomica Hungarica, 49, 2001248DiscussionBoth population diversity and fungal abundance were affected by leafposition in the canopy. Fluctuations in temperature and relative humiditydecrease towards lower positions in the canopy (Blakeman, 1985). Andrews(1991) suggested the importance of UV light intensity in the regulation of fungalpopulations in the canopy. This might explain the lower number of fungirecorded on leaves in the upper part of the canopy in both the ControlGreenhouse and Commercial Greenhouse II. According to Odum (1985)biodiversity is low in physically controlled ecosystems, i.e. in those subjected tostrongly limited physical factors. Leaves located within the canopy mightprovide a less restricted environment, thus resulting in more diversecommunities. Based on the Control Greenhouse and Commercial Greenhouse II,it was concluded that the fungal population was more abundant on lower leaves,while medium and upper leaves had similar fungal populations. According toAndrews (1991) the presence of abundant nutrient sources in lower leaves due tothe proximity of the soil might also explain the presence of higher numbers offungi. On the other hand, Ruinen (1961) asserted that the successional climax ofphylloplane microbial populations could not be reached until the leaves reachedmaturity, and the lower leaves are the most mature ones on the plant. Thesefindings regarding abundance and diversity and their association with positionwithin the canopy, confirm the results obtained for the Control Greenhouse andCommercial Greenhouse II, but not those found for Commercial Greenhouse I,where the upper leaves had more developed fungal populations than any of theother sprayed leaves. The longer time which elapsed (30 days) from the lastapplication in Commercial Greenhouse I might explain this phenomenon. It isimportant to consider both abundance and diversity when planning naturalbiological control (Cook and Baker, 1989). So the abundance and diversity ofsaprophytic fungi achieved in the Control Greenhouse might be more suitablefor appropriate disease suppression.The qualitative composition of the fungal population was also affected bythe fungicide application scheme and leaf position in the canopy. Most of theyeasts and fungi found in the upper leaves belong to the naturally-occurringresident microorganisms on aerial plant surfaces listed by Blakeman andFokkema (1982). The phyllosphere of the lower leaves is inhabited by a numberof soil fungi, mainly Trichoderma spp. and Chaetomium globosum. Theubiquitous genera Aspergillus and Penicillium have a distribution profileunrelated with leaf position in the canopy. The effect of the fungicide applicationscheme on the saprophytic fungus composition allows a distinction to be madebetween fungicide-tolerant species, i.e. those that prevailed and persisted on theleaves from the commercial greenhouses, and susceptible species, which werepresent only in the Control Greenhouse. The results demonstrated that fungicideapplication led to the suppression of many potential antagonists of foliarSAPROPHYTIC FUNGI ON TOMATO PHYLLOPLANEActa Agronomica Hungarica, 49, 2001249pathogens. Resident yeasts, described by many authors as effective biocontrolagents in the phylloplane (Suzzi et al., 1995; Fokkema and Van der Meulen,1976) were the most affected by fungicide application. The same was valid forTrichoderma spp., an acknowledged biocontrol agent whose population wasparticularly affected by fungicide applications.The prevalence of Aspergillus spp. and Penicillium spp. on leaves fromCommercial Greenhouses I and II suggests that fungicide resistance may be theresult of the selection pressure performed on both genera (Bollen, 1971). Thecapacity of some indigenous saprobic phylloplane fungi to develop resistance tofungicides is a desirable characteristic which could be utilised in biocontrolmethods (Fokkema and Naaj, 1981; Fokkema, 1993).AcknowledgementsThe authors wish to thank Dr. Pedro Balatti and Mr. M. R. Simón for their critical readingof the manuscript.ReferencesAndrews, J., Kennerly, C. (1978): The effect of a pesticide program on non target epiphyticmicrobial populations of apple leaves. Canadian Journal of Microbiology, 24, 1058–1072.Andrews, J. (1991): Biological control in the phyllosphere. Annual Review of Phytopathology, 30,603–635.Baker, R. (1991): Diversity in biological control. Crop Protection, 10, 85–94.Blakeman, J., Fokkema, N. (1982): Potential for biological control of plant diseases on thephylloplane. Annual Review of Phytopathology, 20, 167–192Blakeman, J. (1985): Ecological succession of leaf surface microorganisms in relation tobiological control. pp. 63–82. In: Windels, C., Lindow, S. (eds.), Biological Control in thePhylloplane. APS Press, Saint Paul, USA.Bollen, G. (1971): Resistance to benomyl and some chemically related compounds in strains ofPenicillium species. Netherland Journal of Plant Pathology, 77, 187–193.Cook, R., Baker, K. (1989): The Nature and Practice of Biological Control of Plant Pathogens.APS Press, Saint Paul, USA. 539 p.De Waard, M., Georgepoulos, S., Hollomond, D., Ishii, D., Leroux, P., Ragsdale, N., Shwinn, C.(1993): Chemical control of plant diseases: Problems and prospects. Annual Review ofPhytopathology, 31, 403–421.Fokkema, N., Van der Meulen, F. (1976): Antagonism of yeastlike phyllosphere against Septorianodorum on wheat leaves. Netherland Journal of Plant Pathology, 82, 13–16.Fokkema, N., Naaj, M. (1981): The effect of fungicides on the microbial balance in thephyllosphere. EPPO Bulletin, 11, 303–310.Fokkema, N. (1993): Opportunities and problems of control of foliar pathogens withmicroorganisms. Pesticides Science, 37, 411–416.Marois, J., Coleman, P. (1994): Ecological succession and biological control in the phyllosphere.Canadian Journal of Botany, 73, (Suppl 1) S76–S82.Mónaco, C., Nico, A., Mitidieri, I., Alippi, H. (1999): Saprobic fungi inhabiting tomatophylloplane as possible antagonists of Alternaria solani. Acta Agronomica Hungarica, 47,397–403.C. I. MÓNACO et al.Acta Agronomica Hungarica, 49, 2001250Morris, C., Rouse, D. (1991): Role of nutrients in regulating epiphytic bacterial populations. pp.63–82. In: Windels, C., Lindow, S. (eds.), Biological Control in the Phylloplane. APSPress, Saint Paul, USA.Odum, E. (1985): Ecología. 3rd Edn. Interamericana, México DF. 405 p.Ruinen, J. (1961): The phyllosphere: An ecologically neglected milieu. Plant and Soil, 15, 81–109.Suzzi, G., Romano, P., Ponti, I., Montuschi, C. (1995): Natural wine yeasts as biocontrol agents.Journal of Applied Bacteriology, 78, 304–308.

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Saprophytic fungi on tomato phylloplane: Effect of fungicides and leaf position on abundance, composition and diversity

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