Microb Ecol (1992) 23:41-52
MICROBIAL ECOLOGY © Springer-VerlagNew York Inc. 1992
Influence of Climatic and Nutritional Factors on Yeast Population Dynamics in the Phyllosphere of Wheat Aleid J. Dik, ~,2,* Nyckle J. F o k k e m a , 3 a n d J o h a n n e s A. v a n Pelt 1 ~Department of Plant Ecology and Evolutionary Biology, Section of Plant Pathology, University of Utrecht, P.O. Box 80084, NL-3508 TB Utrecht; 2Department of Theoretical Production Ecology, Wageningen Agricultural University, P.O. Box 430, NL-6700 AK Wageningen; and, 3Research Institute for Plant Protection, P.O. Box 9060, NL-6700 GW Wageningen, The Netherlands Received."May 13, 1991; Revised."August 19, 1991
Abstract. T h e role o f saprophytic phyllosphere yeasts in r e m o v i n g a p h i d h o n e y d e w and other nutrients f r o m wheat leaves was evaluated in growth cabinet e x p e r i m e n t s at different t e m p e r a t u r e s a n d relative humidities. P o p ulation densities o f b o t h p i n k a n d white yeasts (Sporobolomyces roseus a n d Cryptococcus laurentiL respectively) increased between 12 a n d 24°C, if nutrients were supplied. W h i t e yeast n u m b e r s increased rapidly at a constant v a p o r pressure deficit (VPD) o f 0. l0 k P a a n d alternating V P D s o f 0.10 a n d 0.61 k P a (each 12 hours per day) but decreased at a constant V P D o f 0.61 kPa. In growth cabinet e x p e r i m e n t s with aphids on wheat plants, the a m o u n t o f a p h i d h o n e y d e w on the leaves was lower w h e n yeast p o p u l a t i o n densities were high. A d d i t i o n o f a m i n o acids to leaves with h o n e y d e w h a d no effect on yeast p o p u l a t i o n density or the rate o f h o n e y d e w c o n s u m p t i o n . This indicated that low concentrations o f a m i n o acids in a p h i d h o n e y d e w are not a limiting factor for h o n e y d e w c o n s u m p t i o n b y the yeasts. T h e naturally occurring saprophytes efficiently r e m o v e d fructose, sucrose, a n d melezitose f r o m the phyllosphere o f field-grown wheat plants.
Introduction P i n k a n d white yeasts (mainly Sporobolomyces spp. a n d Cryptococcus spp., respectively) p r e d o m i n a t e in the saprophytic phyllosphere c o m m u n i t i e s on wheat leaves. T h e yeasts can use a p h i d honeydew, pollen, and leaf exudates as a nutrient source. In a controlled e n v i r o n m e n t , yeasts can efficiently r e m o v e exogenous nutrients f r o m w h e a t leaves [6]. In field experiments, reduction o f * Present address: Department of Plant Pathology, University of Wisconsin, 1630 Linden Drive, Madison, Wisconsin 53706, USA. Offprint requests to: Aleid Josine Dik, Department of Plant Pathology, University of Wisconsin, 1630 Linden Drive, Madison, Wisconsin 53706, USA.
42
A.J. Dik et al.
the naturally occurring yeast population by broad-spectrum fungicides enhanced the accumulation of aphid honeydew, which is detrimental to yield, relative to untreated plots [5]. This paper describes the results of experiments done to provide additional information about effects of climatic conditions and substrate composition on yeast population dynamics, which is needed to evaluate the ecological role of the yeasts in removal of nutrients from the phyllosphere. Although Bashi and Fokkema [2] studied the influence of relative humidity on Sporobolomyces roseus, little information is available on the influence of this factor on Cryptococcus spp. or of temperature on both pink and white yeasts. The composition of aphid honeydew has been reviewed by Auclair [1 ]. The most common sugars are fructose, glucose, and sucrose; others include maltose, melezitose, and some unidentified oligosaccharides. Honeydew on field-grown winter wheat, infested with the aphids Sitobion avenae and Metopolophium dirhodum, was shown to contain fructose, maltose, trehalose, and melezitose, and honeydew from S. avenae reared on spring wheat in a growth cabinet contained fructose, sucrose, and trehalose (A. J. Dik, unpublished results). Both Sporobolomyces spp. and Cryptococcus spp. can consume these carbon compounds [8, 9; A. J. Dik, unpublished results]. However, although some amino acids are present in aphid honeydew [1, 10], the C/N-ratio is high [5] and amino acids might be a limiting factor for honeydew consumption by the yeasts. This paper describes growth cabinet experiments on the influence of VPD on white yeasts and of temperature on pink and white yeasts. Furthermore, experiments are described with aphids on spring wheat, investigating the influence of additional amino acids on honeydew consumption by yeasts. Finally, the consumption of fructose, sucrose, and melezitose by naturally occurring yeasts and other saprophytes on field-grown wheat was determined.
Materials and Methods
Plants Spring wheat, Triticurn aestivum L. (cv. Minaret), was grown in potting soil (Triomf nr. 17, TRIO B.V.) in pots of 20 x 20 cm (25 seeds per pot) in a greenhouse at temperatures between 18 and 25°C. From October to May, additional light was provided for 16 hours per day by high-pressure sodium lamps (Philips, SON-T, 400 W). If necessary, mildew infection, caused by Erysiphe graminis f.sp. triticL was suppressed by spraying with ethirimol (Milgo E, 2.5 ml liter -~, I.C.I.), avoiding the flag leaves. Ethirimol has no effect on yeasts [3]. For the field experiment, wheat was sown in a humous sandy soil in the botanical garden, Cantonspark, in Baarn. To prevent interference of pollen deposition with the experiments, the plants were used before flowering (growth stage [G.S.] 50) [111.
Yeasts The pink yeast Sporobolomyces roseus (isolate WCS30) and the white yeast Cryptococcus laurentii (isolate WCS50) were grown at 20°C on potato dextrose agar (Difco, 39 g liter-~). After 5-7 days, separate suspensions were made by scraping the cultures cautiously and suspending the yeasts in a sterile Tween 80 (0.01%) solution. In all experiments, either pink or white yeasts were applied,
Climatic and Nutritional Factors and Yeasts
43
except for one of the experiments with aphids, in which a mixture of yeasts was applied. The yeast suspension was filtered through glass wool and the concentration of yeast cells was adjusted to a concentration of 1-2.5 x 106 cells ml-% using a hemocytometer. In treatments with nutrients, the nutrients were added to yeast suspensions before spraying. In two of the experiments with aphids, yeasts were also applied at a concentration of 107 cells ml-L Yeasts were sprayed on the leaves until run-offwith a glass reagent sprayer (Desaga), and were sprayed on both sides of the flag leaves in experiments with aphids and on the upper side of the flag leaf in all others.
Aphids Aphids of the species S i t o b i o n a v e n a e were reared on spring wheat plants cv. Minaret (G.S. 45) [11], in a growth cabinet at 22°C and 70% relative humidity (r.h.) during a 16 hour light period and 18°C and 95% r.h. during the 8 hour dark period. The aphids were provided with fresh plants every two weeks.
Assessment of Carbohydrates and of Population Densities of Saprophytes Flag leaves (eight per treatment in the experiments with aphids; six per treatment in the other growth cabinet experiments; six per replicate with four replicates per treatment in the field experiment) were sampled at regular intervals by cutting the stem ca. 10 cm below the flag leaf. For growth cabinet experiments, each leaf was placed in a 300 ml Erlenmeyer flask, containing 100 ml sterile Tween 80 solution (0.01%). In the field experiment, six leaves were placed in a 1,000-ml Erlenmeyer flask, containing 1,000 ml sterile Tween 80 solution (0.01%). The leaves were submerged, hut the cut edges were above the fluid to avoid leakage of assimilates. The flasks were treated in an ultrasonic bath (Branson, 50,000 Hz) for five minutes in order to separate nutrients and yeast cells from the leaves. Afterwards, the area of each leaf was measured with an automatic area meter (Hayashi Denkoh). Yeast and ftmgal population densities were assessed by plating samples of 100 #l on basal yeast agar [bya, containing 20 g glucose, 1 g yeast extract (Difco), 10 g proteose pepton (Difco), 15 g agar (Bitek), 106 i.u. streptomycin sulphate + 106 i.u. sodium benzylpenicillin (Pharmachemie) per liter]. Bacterial population densities were assessed by plating samples of 50/~l on tryptic soy agar (tsa, Difco). All plating was done in triplicate after appropriate dilution. Colonies were counted after incubation at 21°C for 2-3 days (tsa) or 5-7 days (bya), and population density for each species was calculated and expressed as colony forming units (cfu) per cm 2 of leaf area. The relative growth rate of the populations was calculated for each interval between sampling days. To assess the carbohydrate concentration on the leaves, a sample of ca. 5 ml of the leaf washings was filtered through a 0.2/~m membrane filter (Sartorius), and 0.5 ml of the filtered solution was used to assess the carbohydrate concentration colorimetrically with anthrone reagent [7]. The amount of sugars was expressed in ~g glucose per cm 2 of leaf area.
Influence of Climatic Conditions Flag leaves of plants were sprayed with a suspension o f pink or white yeasts (106 cfu ml-t). In all experiments, three different treatments were applied, viz. yeasts without added nutrients, yeasts + 20 g sucrose liter -~ + 5 g yeast extract liter ~, and yeasts + 150 g sucrose liter -~ + 5 g yeast extract liter-L Nutrients were added before the yeasts were applied. The plants were placed in growth cabinets and samples were taken at intervals of 1-3 days. The influence of temperature was studied at 12, 15, 18, 21, and 24°C for white yeasts and 12, 15, 18, and 23°C for pink yeasts. Relative humidity (r.h.) was 70% during the 12 hour light period and 95% during the 12 hour dark period, except for the experiment at 15°C with white yeasts in which the r.h. was 64% during the light period and 94% during the dark period.
44
A.J. Dik et al. r
Nutrients (IJg glucose cm -2)
White yeasts (CFU cm "2)
10
120
A
B
+ _
12o 15 o
10
10
6
100
80
5
,./
/
-B--
18 o
::
21 o
-~-
24 o
", 60
lo
41 o /~-~
~
1°.I
40
15 o
21 o
20
240
10 2 /
i
i
i
i
i
0
2
4
6
8
10
Time (d)
i
i
i
i
i
2
4
6
8
10
12
Time (d)
Fig. 1. Influence of temperature (°C) on A population development of white yeasts and B consumption of nutrients applied as 20 g sucrose + 5 g yeast extract liter -1. The influence of r.h. on white yeasts was studied at 15 and 18°C in a 12 hour light period. Relative humidities were chosen to create vapor pressure deficits (VPD) of 0.10 and 0.61 kPa at both temperatures. A VPD of 0.10 kPa corresponds with 95% r.h. at 18°C and with 94% r.h. at 15°C. A VPD of 0.61 kPa corresponds with 70% r.h. at 18°C and with 64% r.h. at 15°C. Both constant and alternating regimes were studied. In the alternating regimes, each VPD was maintained for 12 hours per day.
Consumption of Aphid Honeydew The consumption of aphid honeydew by yeasts was studied in growth cabinet experiments. Ten wingless adult aphids per pot were put on the plants after emergence of the flag leaf (G.S. 45) [11]. After 14 days, different treatments were applied, each to the flag leaves of all the plants in two pots. The day of application of the treatments was considered to be Day 0. In both experiments, one treatment consisted of spraying with maneb (Maneb 80, 4 g liter J, Akzo) to reduce yeast population densities. In the other treatments, yeasts were applied with or without a mixture of amino acids, and in the experiment with pink yeasts and with a mixture of yeasts, yeasts were applied in different concentrations. The amino acid mixture consisted of DL-alanine (0.12 g liter-I), L-asparagine (0.25 g liter-I), L-glutamine (0.20 g liter-J), glycine (0.07 g liter-i), L-histidine (0.31 g liter-i), L-isoleucine (0.14 g liter-]), DL-leucine (0.23 g liter-l), L-lysine (0.08 g liter-l), L-proline (0.25 g liter-l), L-phenylalanine (0.20 g liter-I), DL-threonine (0.84 g liter-l), L-tyrosine (0.23 g l i t e r 1), and DL-valine (0.25 g liter- 1) (B.D.H., Merck, Sigma). This composition and concentration are the same as determined by HPLC in honeydew of the aphid Sitobion avenae reared for three days in a controlled environment on field-grown winter wheat plants cv. Arminda [10]. One experiment was run with white yeasts, one with pink yeasts, and one with a mixture of pink and white yeasts. Chlortetracycline (Sigma, 100 mg liter-l) was added to all treatments to reduce bacterial populations on the leaves. The plants were incubated in a growth cabinet with a 12 hour light period and a r.h. of 70% during the light period and 95% during the dark period. In the experiment with white yeasts, the temperature was 21°C during the light period and 18°C during the dark period. In the experiments with pink yeasts, and with a mixture of pink and white yeasts, the temperature was 19.5°C continuously. Because aphids can shoot honeydew droplets to neighboring
Climatic and Nutritional Factors and Yeasts
45
White yeasts (CFU cm "2)
Nutrients (IJg glucose cm -2 )
10
500
A
B
-i--
12 o 150
o
400
10
o t° 300
10
4
200
10
~10 a
15 o
-~-
18 o
~-
210
100
240
lo2~ 0
i
i
i
i
i
2
4
6
S
10
12
0L 0
= 2
= 4
Time (d)
i 6
8
10
12
Time (d)
Fig. 2. Influence of temperature (°C) on A population development of white yeasts and B consumption of nutrients applied as 150 g sucrose + 5 g yeast extract liter -1. Nutrients (pg glucose cm -2 )
Pink yeasts (CFU cm -2) 10
120
A
D
~
12 o
--,~- 15 o 10 e
100
~
8060
- E - 18 o
~ ~
--"
MICROBIAL ECOLOGY © Springer-VerlagNew York Inc. 1992
Influence of Climatic and Nutritional Factors on Yeast Population Dynamics in the Phyllosphere of Wheat Aleid J. Dik, ~,2,* Nyckle J. F o k k e m a , 3 a n d J o h a n n e s A. v a n Pelt 1 ~Department of Plant Ecology and Evolutionary Biology, Section of Plant Pathology, University of Utrecht, P.O. Box 80084, NL-3508 TB Utrecht; 2Department of Theoretical Production Ecology, Wageningen Agricultural University, P.O. Box 430, NL-6700 AK Wageningen; and, 3Research Institute for Plant Protection, P.O. Box 9060, NL-6700 GW Wageningen, The Netherlands Received."May 13, 1991; Revised."August 19, 1991
Abstract. T h e role o f saprophytic phyllosphere yeasts in r e m o v i n g a p h i d h o n e y d e w and other nutrients f r o m wheat leaves was evaluated in growth cabinet e x p e r i m e n t s at different t e m p e r a t u r e s a n d relative humidities. P o p ulation densities o f b o t h p i n k a n d white yeasts (Sporobolomyces roseus a n d Cryptococcus laurentiL respectively) increased between 12 a n d 24°C, if nutrients were supplied. W h i t e yeast n u m b e r s increased rapidly at a constant v a p o r pressure deficit (VPD) o f 0. l0 k P a a n d alternating V P D s o f 0.10 a n d 0.61 k P a (each 12 hours per day) but decreased at a constant V P D o f 0.61 kPa. In growth cabinet e x p e r i m e n t s with aphids on wheat plants, the a m o u n t o f a p h i d h o n e y d e w on the leaves was lower w h e n yeast p o p u l a t i o n densities were high. A d d i t i o n o f a m i n o acids to leaves with h o n e y d e w h a d no effect on yeast p o p u l a t i o n density or the rate o f h o n e y d e w c o n s u m p t i o n . This indicated that low concentrations o f a m i n o acids in a p h i d h o n e y d e w are not a limiting factor for h o n e y d e w c o n s u m p t i o n b y the yeasts. T h e naturally occurring saprophytes efficiently r e m o v e d fructose, sucrose, a n d melezitose f r o m the phyllosphere o f field-grown wheat plants.
Introduction P i n k a n d white yeasts (mainly Sporobolomyces spp. a n d Cryptococcus spp., respectively) p r e d o m i n a t e in the saprophytic phyllosphere c o m m u n i t i e s on wheat leaves. T h e yeasts can use a p h i d honeydew, pollen, and leaf exudates as a nutrient source. In a controlled e n v i r o n m e n t , yeasts can efficiently r e m o v e exogenous nutrients f r o m w h e a t leaves [6]. In field experiments, reduction o f * Present address: Department of Plant Pathology, University of Wisconsin, 1630 Linden Drive, Madison, Wisconsin 53706, USA. Offprint requests to: Aleid Josine Dik, Department of Plant Pathology, University of Wisconsin, 1630 Linden Drive, Madison, Wisconsin 53706, USA.
42
A.J. Dik et al.
the naturally occurring yeast population by broad-spectrum fungicides enhanced the accumulation of aphid honeydew, which is detrimental to yield, relative to untreated plots [5]. This paper describes the results of experiments done to provide additional information about effects of climatic conditions and substrate composition on yeast population dynamics, which is needed to evaluate the ecological role of the yeasts in removal of nutrients from the phyllosphere. Although Bashi and Fokkema [2] studied the influence of relative humidity on Sporobolomyces roseus, little information is available on the influence of this factor on Cryptococcus spp. or of temperature on both pink and white yeasts. The composition of aphid honeydew has been reviewed by Auclair [1 ]. The most common sugars are fructose, glucose, and sucrose; others include maltose, melezitose, and some unidentified oligosaccharides. Honeydew on field-grown winter wheat, infested with the aphids Sitobion avenae and Metopolophium dirhodum, was shown to contain fructose, maltose, trehalose, and melezitose, and honeydew from S. avenae reared on spring wheat in a growth cabinet contained fructose, sucrose, and trehalose (A. J. Dik, unpublished results). Both Sporobolomyces spp. and Cryptococcus spp. can consume these carbon compounds [8, 9; A. J. Dik, unpublished results]. However, although some amino acids are present in aphid honeydew [1, 10], the C/N-ratio is high [5] and amino acids might be a limiting factor for honeydew consumption by the yeasts. This paper describes growth cabinet experiments on the influence of VPD on white yeasts and of temperature on pink and white yeasts. Furthermore, experiments are described with aphids on spring wheat, investigating the influence of additional amino acids on honeydew consumption by yeasts. Finally, the consumption of fructose, sucrose, and melezitose by naturally occurring yeasts and other saprophytes on field-grown wheat was determined.
Materials and Methods
Plants Spring wheat, Triticurn aestivum L. (cv. Minaret), was grown in potting soil (Triomf nr. 17, TRIO B.V.) in pots of 20 x 20 cm (25 seeds per pot) in a greenhouse at temperatures between 18 and 25°C. From October to May, additional light was provided for 16 hours per day by high-pressure sodium lamps (Philips, SON-T, 400 W). If necessary, mildew infection, caused by Erysiphe graminis f.sp. triticL was suppressed by spraying with ethirimol (Milgo E, 2.5 ml liter -~, I.C.I.), avoiding the flag leaves. Ethirimol has no effect on yeasts [3]. For the field experiment, wheat was sown in a humous sandy soil in the botanical garden, Cantonspark, in Baarn. To prevent interference of pollen deposition with the experiments, the plants were used before flowering (growth stage [G.S.] 50) [111.
Yeasts The pink yeast Sporobolomyces roseus (isolate WCS30) and the white yeast Cryptococcus laurentii (isolate WCS50) were grown at 20°C on potato dextrose agar (Difco, 39 g liter-~). After 5-7 days, separate suspensions were made by scraping the cultures cautiously and suspending the yeasts in a sterile Tween 80 (0.01%) solution. In all experiments, either pink or white yeasts were applied,
Climatic and Nutritional Factors and Yeasts
43
except for one of the experiments with aphids, in which a mixture of yeasts was applied. The yeast suspension was filtered through glass wool and the concentration of yeast cells was adjusted to a concentration of 1-2.5 x 106 cells ml-% using a hemocytometer. In treatments with nutrients, the nutrients were added to yeast suspensions before spraying. In two of the experiments with aphids, yeasts were also applied at a concentration of 107 cells ml-L Yeasts were sprayed on the leaves until run-offwith a glass reagent sprayer (Desaga), and were sprayed on both sides of the flag leaves in experiments with aphids and on the upper side of the flag leaf in all others.
Aphids Aphids of the species S i t o b i o n a v e n a e were reared on spring wheat plants cv. Minaret (G.S. 45) [11], in a growth cabinet at 22°C and 70% relative humidity (r.h.) during a 16 hour light period and 18°C and 95% r.h. during the 8 hour dark period. The aphids were provided with fresh plants every two weeks.
Assessment of Carbohydrates and of Population Densities of Saprophytes Flag leaves (eight per treatment in the experiments with aphids; six per treatment in the other growth cabinet experiments; six per replicate with four replicates per treatment in the field experiment) were sampled at regular intervals by cutting the stem ca. 10 cm below the flag leaf. For growth cabinet experiments, each leaf was placed in a 300 ml Erlenmeyer flask, containing 100 ml sterile Tween 80 solution (0.01%). In the field experiment, six leaves were placed in a 1,000-ml Erlenmeyer flask, containing 1,000 ml sterile Tween 80 solution (0.01%). The leaves were submerged, hut the cut edges were above the fluid to avoid leakage of assimilates. The flasks were treated in an ultrasonic bath (Branson, 50,000 Hz) for five minutes in order to separate nutrients and yeast cells from the leaves. Afterwards, the area of each leaf was measured with an automatic area meter (Hayashi Denkoh). Yeast and ftmgal population densities were assessed by plating samples of 100 #l on basal yeast agar [bya, containing 20 g glucose, 1 g yeast extract (Difco), 10 g proteose pepton (Difco), 15 g agar (Bitek), 106 i.u. streptomycin sulphate + 106 i.u. sodium benzylpenicillin (Pharmachemie) per liter]. Bacterial population densities were assessed by plating samples of 50/~l on tryptic soy agar (tsa, Difco). All plating was done in triplicate after appropriate dilution. Colonies were counted after incubation at 21°C for 2-3 days (tsa) or 5-7 days (bya), and population density for each species was calculated and expressed as colony forming units (cfu) per cm 2 of leaf area. The relative growth rate of the populations was calculated for each interval between sampling days. To assess the carbohydrate concentration on the leaves, a sample of ca. 5 ml of the leaf washings was filtered through a 0.2/~m membrane filter (Sartorius), and 0.5 ml of the filtered solution was used to assess the carbohydrate concentration colorimetrically with anthrone reagent [7]. The amount of sugars was expressed in ~g glucose per cm 2 of leaf area.
Influence of Climatic Conditions Flag leaves of plants were sprayed with a suspension o f pink or white yeasts (106 cfu ml-t). In all experiments, three different treatments were applied, viz. yeasts without added nutrients, yeasts + 20 g sucrose liter -~ + 5 g yeast extract liter ~, and yeasts + 150 g sucrose liter -~ + 5 g yeast extract liter-L Nutrients were added before the yeasts were applied. The plants were placed in growth cabinets and samples were taken at intervals of 1-3 days. The influence of temperature was studied at 12, 15, 18, 21, and 24°C for white yeasts and 12, 15, 18, and 23°C for pink yeasts. Relative humidity (r.h.) was 70% during the 12 hour light period and 95% during the 12 hour dark period, except for the experiment at 15°C with white yeasts in which the r.h. was 64% during the light period and 94% during the dark period.
44
A.J. Dik et al. r
Nutrients (IJg glucose cm -2)
White yeasts (CFU cm "2)
10
120
A
B
+ _
12o 15 o
10
10
6
100
80
5
,./
/
-B--
18 o
::
21 o
-~-
24 o
", 60
lo
41 o /~-~
~
1°.I
40
15 o
21 o
20
240
10 2 /
i
i
i
i
i
0
2
4
6
8
10
Time (d)
i
i
i
i
i
2
4
6
8
10
12
Time (d)
Fig. 1. Influence of temperature (°C) on A population development of white yeasts and B consumption of nutrients applied as 20 g sucrose + 5 g yeast extract liter -1. The influence of r.h. on white yeasts was studied at 15 and 18°C in a 12 hour light period. Relative humidities were chosen to create vapor pressure deficits (VPD) of 0.10 and 0.61 kPa at both temperatures. A VPD of 0.10 kPa corresponds with 95% r.h. at 18°C and with 94% r.h. at 15°C. A VPD of 0.61 kPa corresponds with 70% r.h. at 18°C and with 64% r.h. at 15°C. Both constant and alternating regimes were studied. In the alternating regimes, each VPD was maintained for 12 hours per day.
Consumption of Aphid Honeydew The consumption of aphid honeydew by yeasts was studied in growth cabinet experiments. Ten wingless adult aphids per pot were put on the plants after emergence of the flag leaf (G.S. 45) [11]. After 14 days, different treatments were applied, each to the flag leaves of all the plants in two pots. The day of application of the treatments was considered to be Day 0. In both experiments, one treatment consisted of spraying with maneb (Maneb 80, 4 g liter J, Akzo) to reduce yeast population densities. In the other treatments, yeasts were applied with or without a mixture of amino acids, and in the experiment with pink yeasts and with a mixture of yeasts, yeasts were applied in different concentrations. The amino acid mixture consisted of DL-alanine (0.12 g liter-I), L-asparagine (0.25 g liter-I), L-glutamine (0.20 g liter-J), glycine (0.07 g liter-i), L-histidine (0.31 g liter-i), L-isoleucine (0.14 g liter-]), DL-leucine (0.23 g liter-l), L-lysine (0.08 g liter-l), L-proline (0.25 g liter-l), L-phenylalanine (0.20 g liter-I), DL-threonine (0.84 g liter-l), L-tyrosine (0.23 g l i t e r 1), and DL-valine (0.25 g liter- 1) (B.D.H., Merck, Sigma). This composition and concentration are the same as determined by HPLC in honeydew of the aphid Sitobion avenae reared for three days in a controlled environment on field-grown winter wheat plants cv. Arminda [10]. One experiment was run with white yeasts, one with pink yeasts, and one with a mixture of pink and white yeasts. Chlortetracycline (Sigma, 100 mg liter-l) was added to all treatments to reduce bacterial populations on the leaves. The plants were incubated in a growth cabinet with a 12 hour light period and a r.h. of 70% during the light period and 95% during the dark period. In the experiment with white yeasts, the temperature was 21°C during the light period and 18°C during the dark period. In the experiments with pink yeasts, and with a mixture of pink and white yeasts, the temperature was 19.5°C continuously. Because aphids can shoot honeydew droplets to neighboring
Climatic and Nutritional Factors and Yeasts
45
White yeasts (CFU cm "2)
Nutrients (IJg glucose cm -2 )
10
500
A
B
-i--
12 o 150
o
400
10
o t° 300
10
4
200
10
~10 a
15 o
-~-
18 o
~-
210
100
240
lo2~ 0
i
i
i
i
i
2
4
6
S
10
12
0L 0
= 2
= 4
Time (d)
i 6
8
10
12
Time (d)
Fig. 2. Influence of temperature (°C) on A population development of white yeasts and B consumption of nutrients applied as 150 g sucrose + 5 g yeast extract liter -1. Nutrients (pg glucose cm -2 )
Pink yeasts (CFU cm -2) 10
120
A
D
~
12 o
--,~- 15 o 10 e
100
~
8060
- E - 18 o
~ ~
--"
