Plant Pathology (2003) 52, 546–552
Effects of temperature, relative humidity and duration of wetness period on germination and infection by conidia of the pear scab pathogen (Venturia nashicola) Blackwell Publishing Ltd.
B. Lia, H. Zhaoa, B. Lia and X.-M. Xub*† a
Department of Plant Protection, Laiyang Agricultural College, 65 Wenhua Road, Laiyang 265200, Shandong Province, China; and Horticulture Research International, East Malling, West Malling, Kent ME 19 6BJ, UK
b
Experiments were conducted to determine the effects of temperature, relative humidity (RH) and duration of wetness period on in vitro germination of conidia and infection of detached pear leaves by Venturia nashicola, the causal agent of pear scab. Conidia germinated only in near-saturation humidity (RH > 97%). The final percentage germination (24 h after inoculation) at 100% RH without free water was less than half that in free water. Conidia germinated over the range of temperatures tested (5 – 30°C); the optimum temperature for germination was ≈21°C. Changes in percentage germination of conidia over time were fitted by logistic models at each individual temperature. Polynomial models satisfactorily described the relationships between two (rate and time to 50% of maximum germination) of the three logistic model parameters and temperature. The minimum length of the wetness period for successful infection of detached pear leaves by conidia was observed at several temperatures. The shortest length of wetness period required for infection was 7 h at 22°C. Two polynomial models fitted well the relationship between the minimum wetness duration required for infection, and temperature. Keywords: forecasting, germination, infection, model, pear scab
Introduction Pear scab, caused by Venturia nashicola (Tanaka & Yamamoto, 1964), is an economically important disease in China. The pathogen is different from the European pear scab fungus (Venturia pirina). It infects leaves, fruits and young shoots, resulting in significant annual yield loss of pears in China. The pathogen can overwinter in buds on pear trees as dormant mycelia, in dead leaves on the ground as immature pseudothecia, and / or on the surface of twigs or diseased leaves as conidia (Li, 1959; Luo, 1983; Yin & Yu, 1988). Conidia, produced from the overwintered dormant mycelia in buds or / and on the surface of twigs or dead leaves, are considered to be the main form of primary inoculum in spring in most areas of northern China (Li, 1959; Yin & Yu, 1988). The development of a pear scab epidemic is generally similar to that for apple scab, caused by Venturia inaequalis. However, in contrast to apple scab (MacHardy, 1996), there has been limited research on the biology and epidem*To whom correspondence should be addressed. †E-mail:
[email protected] Accepted 11 May 2003
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iology of pear scab. Consequently, in China pear scab is currently controlled mainly by scheduled applications of fungicides. For apple scab, prediction systems have been developed and successfully used in practice to assist in timing fungicide applications (MacHardy, 1989; Xu et al., 1995; MacHardy, 1996; Berrie, 1997). Use of a forecasting system led to a reduction in fungicide inputs while maintaining satisfactory control of apple scab in England (Berrie, 1994; Berrie, 1997). Generally, these apple scab forecasting systems are based on relationships between scab incidence or severity, resulting from infection by conidia and ascospores, with temperature, amount and duration of rainfall, and duration of leaf wetness (Schwabe, 1980; MacHardy, 1989; Xu et al., 1995; MacHardy, 1996). Only limited research has been conducted on the quantitative epidemiology of V. nashicola (Luo, 1983; Umemoto, 1991). Luo (1983) reported that conidia of pear scab can germinate at temperatures from 2 to 30°C, with the optimum between 15 and 20°C, and that free water is essential for conidia to germinate. No conidia germinated at relative humidities (RH) below 95%. Umemoto (1991) reported that increase in the incidence of leaf infection by conidia during wetness periods was greatest at 15 and 20°C after an initial 9 h wet period. There were no scab symptoms observed at 30°C after 36 h of leaf wetting. Currently © 2003 BSPP
Conidial germination and infection by Venturia nashicola
there are not sufficient quantitative data to develop a forecasting model for pear scab in China. The research described here aimed to obtain quantitative data on the effects of environmental conditions on development of V. nashicola, specifically (i) the effects of temperature and RH on in vitro germination of conidia; (ii) the effects of temperature on the temporal dynamics of in vitro germination of conidia in free water; and (iii) the effects of temperature and duration of wetness period on infection of detached pear leaves by conidia. Regression models were developed to describe the observed effects, with a view to the development of a pear scab forecasting system for use in integrated disease management strategies in China.
Materials and methods Inoculum Scab lesions from diseased leaves of pear cv. Lai-yang-ci-pear (Pyrus bretschneideri), grown in a commercial orchard of Laiyang Agriculture College, were collected when needed and washed in distilled water to make a conidial suspension. Experiments were conducted during the period 4 May to 4 August 2002. On the day of each experiment, three leaves with fresh, sporulating scab lesions were collected from the orchard and washed together to make a spore suspension, hence the resulting suspension was a mixed inoculum. For all the experiments inoculum suspensions were made from leaves collected from the same orchard. The final suspensions were adjusted, using a haemocytometer, to concentrations of c. 5 × 104 conidia mL−1 for in vitro germination tests and c. 1 × 106 conidia mL−1 for infection experiments on detached leaves.
Effect of temperature and RH on in vitro conidial germination Germination of conidia was observed at six temperatures (5, 10, 15, 20, 25 and 30°C), at each of four RH levels (100, 99, 97 and 95%). In addition, a free-water treatment was included at each temperature. In total there were 30 treatments. Relative humidities of 100, 99, 97 and 95% were obtained by amending water agar (Qing-dao Agar Powder Factory, China) with 0, 0·3, 0·9 and 1·5 m NaCl, respectively, inside sealed Petri dishes (Lang, 1967; Harris et al., 1970; Alderman & Beute, 1986; Xu et al., 2001). Two glass slides (26 × 76 mm) were placed on the lid of each Petri dish containing approximately 30 mL water agar amended with the appropriate amount of NaCl to achieve the desired RH. Two 10 µL droplets of conidial suspension were separately placed on each slide using a micropipette and air-dried at room temperature (normally between 15 and 25°C). On average it took ≈20 min from washing conidia off diseased leaves for the inoculum droplets to dry. The plate was then immediately sealed with parafilm (American National Can, Chicago, USA) and placed upside down in an incubator (LRH-250-GII, Guang-dong Medical Instrument Factory, China) set to © 2003 BSPP Plant Pathology (2003) 52, 546–552
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the appropriate temperature. For the free-water treatment, a water agar plate without NaCl (as for the 100% RH treatment) was sealed immediately after the inoculum was placed on the slides, without air-drying. Percentage germination was recorded after 24 h. A drop of cotton blue in lactophenol [lactic acid : phenol : glycerin : distilled water = 1 : 1 : 2 : 1 (v/v/v/ v)] was placed on each inoculum droplet to stop any further germination and preserve the conidia. Percentage germination was estimated by examining 100 conidia in each inoculum droplet under the microscope; a conidium was considered to have germinated when its germination tube was longer than half its length. There was one plate (400 conidia examined for germination) for each treatment, and the experiment was repeated four times. For each replicate experiment, each incubator was randomly assigned to one of the experimental temperatures.
Effect of temperature on temporal dynamics of germination Changes with time in the percentage germination of conidia were studied with free water at six temperatures: 5, 10, 15, 20, 25 and 30°C. Four glass slides (26 × 76 mm) were placed on the lid of a Petri dish containing ≈30 mL water agar to maintain RH at 100% inside the plate. Two 10 µL droplets of conidial suspension were separately placed on each glass slide using a micropipette. Each plate was then immediately sealed with parafilm (inoculum droplets were not dried) and placed upside down in an incubator set to the appropriate temperature. There were 15 plates for each temperature. For the 5°C treatment germination was first assessed 4 h after the inoculum was placed on the glass slide, and thereafter was recorded every 2 h (for a few replicates, germination was not recorded at 18, 20 or 22 h). For temperatures of 10, 15, 20 and 25°C, percentage germination was first assessed at 2 h and thereafter at hourly intervals until 11 or 12 h. At 30°C, germination was assessed at 3 h and thereafter at hourly intervals until 12 h (for a few replicates, additional observations were made at 13, 16 and 20 h). Finally, for all the temperatures germination was also recorded 24 h after inoculation. One plate was removed at each assessment time and a drop of cotton blue in lactophenol was placed on each inoculum droplet to stop further germination of conidia and to preserve them. Percentage germination was estimated by examining 100 conidia in each inoculum droplet under the microscope. The experiment was repeated twice (a total of three replicate experiments at all temperatures), and two additional tests were done at 5 and 30°C because of large variations between replicates at these temperatures. For each replicate experiment each incubator was randomly assigned to one of the experimental temperatures.
Effect of temperature on infection Leaves on detached shoots of cv. Ya-pear trees were used to evaluate the effects of temperature and duration of wetness period on infection by conidia. Five temperatures
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were used: 10, 14, 18, 22 and 26°C. The experimental trees, from which shoots were removed for infection studies, were maintained in a mobile polythene tunnel. The trees were covered with polythene during the night, but not during the day, except when rain was forecast. This was done to keep leaves dry and thus prevent any infection by V. nashicola prior to the inoculation studies. Forty shoots with young, growing leaves were cut from young trees on the day of each experiment, giving eight shoots per temperature. Leaves older than leaf −4 were removed (the youngest unrolled leaf was numbered leaf 0 and leaves at positions −1, −2, −3, etc. were older and increasingly larger than leaf 0, leaf −1 being adjacent to leaf 0). Each shoot was then maintained with its base inside a bottle (500 mL) full of water. The whole shoot was inoculated with a suspension of conidia using a hand sprayer (500 mL) and covered with a polythene bag to keep the inoculated shoot (leaves) wet; the shoots were then placed inside an incubator set to the appropriate temperature. Leaf −1 on each inoculated shoot was tagged at the time of inoculation. One shoot was taken out and air-dried at room temperature (15–25°C) at hourly intervals beginning at 13, 8, 6, 6 and 6 h after inoculation for the 10, 14, 18, 22 and 26°C treatments, respectively. In total there were eight hourly assessments at each temperature. The shoot and bottle were then transferred to an incubator set to 22°C and maintained for a further 96 h. The tagged leaf was collected from each shoot, cleared and stained for microscopic examination. Leaves were cleared and stained using 1% trypan blue in saturated chloral hydrate [CCl3CH(OH)2] solution [chloral hydrate : distilled water = 5 : 2 (w /v)]. Eight discs (15 mm diameter) were taken from each leaf using a cork-borer, and placed in the 1% trypan blue solution for 48 h. After rinsing with distilled water, the discs were transferred into saturated chloral hydrate solution for 24 h, then mounted in glycerol. Ten microscopic view fields at ×200 magnification were randomly selected for each disc; there was at least one conidium within each of the fields. A conidium was considered to have infected the leaf if mycelium was observed immediately under the appressorium. The total number of conidia that had infected the host was recorded. The experiment was performed twice. For each replicate experiment, each incubator was randomly assigned to one of the experimental temperatures.
Results Effects of temperature and RH on in vitro conidial germination anova showed that the main effects of temperature and RH on in vitro germination of V. nashicola conidia were highly significant (P < 0·01). Most variation in germination was caused by RH, which accounted for 70·9% of the total variation on the arcsine scale, compared with 10·5% accounted for by temperature. Conidial germination was greatest in free water, more than double that in the 100% RH treatment. Percentage germination was very low, close to zero, at RH ≤ 97% (Table 1). Percentage germination was greatest at 20°C and lowest at 30°C. For example, germination was only 16·4% under free water at 30°C, compared with 92·9% at 20°C. The interaction between temperature and RH was also significant (P < 0·01), accounting for 8·7% of the total variation. This was mainly the result of the low germination observed in the 5°C/ 100% RH and 10°C /100% RH treatments (Table 1).
Effect of temperature on temporal dynamics of conidial germination
Data analysis anova was used to assess the effects of temperature and RH on the in vitro percentage of conidial germination (p). All percentage germination data were arcsine-transformed (arcsin √p) before anova to stabilize the variance. Logistic models were used to describe the changes with time on conidial germination at each temperature: p = K/{1 + exp[− β(t − M)]}
percentage germination; M is the length of elapsed time (h) until p = K/ 2; and β is the rate (h−1). The rate of epidemic development is of paramount importance for developing a real-time forecasting model. Often the rate parameter used in forecasting models is the logistic rate parameter (β) or the inverse of the time required for a certain process, such as M. The present study therefore used regression analysis to describe the effects of temperature on M and β. Originally the aim of this study was to obtain high quality data on accumulated numbers of conidia that had infected detached leaves over time at each temperature, in order to develop mathematical models describing the observed temporal trends. Unfortunately, this proved to be very difficult. Instead, the observed data were used only to obtain the minimum length (L) of wetness duration required for infection of detached leaves by conidia at each temperature. The relationship between L and temperature was then determined in two ways. In the first (direct) method, L was directly regressed on temperature. In the second (indirect) method, a relationship was first established between L and M, then L was related to temperature via the relationship of M with temperature. genstat (Payne et al., 2000) was used for all statistical analyses.
(1)
where K, M and β are parameters to be estimated and t is the time (h). Parameter K is the maximum possible
Conidial germination was first observed 6, 4, 2, 2, 2 and 3 h after inoculating the slides at 5, 10, 15, 20, 25 and 30°C, respectively, and percentage germination increased rapidly thereafter (Fig. 1). The average percentage germination after 24 h was 73·2, 85·1, 89·8, 91·3, 81·9 and 54·5% at 5, 10, 15, 20, 25 and 30°C, respectively. Percentage germination at 5 and 30°C was highly variable between replicate experiments (Fig. 1). Logistic models satisfactorily described the changes with time in conidial germination at each temperature © 2003 BSPP Plant Pathology (2003) 52, 546–552
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Table 1 Average percentage in vitro germination of Venturia nashicola (based on 2000 conidia) at various combinations of temperature and relative humidity, assessed after 24 h. Relative humidity was controlled by amending water agar with NaCl inside sealed agar plates Temperature (°C) Relative humidity (%)
5 a
Free water 100 99 97 95
55·9 (48·7) 7·2 (13·3) 3·5 (8·7) 1·0 (4·1) 0·1 (0·6)
10
15
20
25
30
81·5 (65·4) 24·1 (28·7) 5·6 (12·4) 1·0 (4·1) 0·3 (1·6)
86·4 (69·0) 47·2 (43·3) 11·1 (17·8) 1·3 (4·5) 0·3 (1·6)
92·9 (75·1) 59·1 (51·0) 16·6 (23·2) 3·5 (9·2) 1·2 (4·6)
78·3 (62·6) 30·3 (32·3) 10·8 (17·4) 0·8 (3·6) 0·3 (1·4)
16·4 (21·4) 7·5 (11·8) 2·9 (7·1) 0·4 (2·1) 0·1 (0·6)
a
Numbers in brackets are averages of arcsine-transformed germination percentages. SEDs on the arcsine scale were 0·97, 1·107 and 2·39 for the relative humidity, temperature and humidity × temperature treatments, respectively.
Table 2 Parameter estimates of logistic models describing the temporal dynamics of in vitro germination of Ventura nashicola conidia at six temperatures. Parameter K is the maximum possible percentage of germination; M is time elapsed until p = K/2; β is the rate (% h−1). These six models are also shown in Fig. 1 Temperature (°C)
K
β
M
Percentage variance accounted for
5 10 15 20 25 30
0·72 ± 0·043 0·82 ± 0·028 0·86 ± 0·019 0·89 ± 0·027 0·81 ± 0·012 0·47 ± 0·042
0·38 ± 0·069 0·90 ± 0·116 1·64 ± 0·225 1·90 ± 0·378 1·59 ± 0·168 0·46 ± 0·106
12·56 ± 0·578 7·34 ± 0·165 4·36 ± 0·097 3·50 ± 0·117 3·54 ± 0·729 7·98 ± 0·651
83·3 94·7 95·8 90·2 96·5 62·0
(Table 2; Fig. 1). Goodness of fit was lowest at 30°C, with 62·0% of variation accounted for by the model. The estimated model parameters differed greatly between temperatures. The estimated maximum percentage germination (K) was similar at 10, 15, 20 and 25°C, and far greater than at 5 or 30°C. The rate of germination (β) increased with temperature above 5°C to a maximum at 20°C, and then decreased with further increases in temperature (Fig. 2a; Table 2), which was the opposite of the relationship between M and temperature (Fig. 2b). The following polynomial equation fitted the relationship between β and temperature (T ), accounting for about 99·3% of the total variation: β = −0·10 + 1·27T − 0·41T 2
3
(2)
where T (°C) was divided by 10 to avoid small values of parameter estimates. The standard errors of the three parameter estimates, K, M and β, were 0·05, 0·05 and 0·02, respectively. Figure 2a shows the fitted model together with the observed values. The relationship between M and T was fitted by the following polynomial model (Fig. 2b): M = 17·50 − 10·55T + 0·82T 3
(3)
where T (°C) was divided by 10 to avoid small values of parameter estimates. The standard errors of the three parameter estimates K, M and β were 0·70, 0·66 and 0·06, respectively. This model accounted for ≈98·9% of the total variation. The estimated optimum temperature from Eqns 2 and 3 was ≈20·7°C. © 2003 BSPP Plant Pathology (2003) 52, 546–552
Effect of temperature on infection by conidia Mycelia were clearly observed in the cleared and stained tissues of inoculated leaves, using light microscopy. Infection by conidia was observed in 29 of the 40 treatment combinations (Table 3). The minimum wetness durations (L) required for successful infection by conidia were 16, 10, 8, 7 and 8 h at 10, 14, 18, 22 and 26°C, respectively. A regression model of L on T was derived directly from the observed data on infection of detached leaves: L = 31·65 − 17·26T + 1·22T 3
(4)
where T (°C) was divided by 10 to avoid small values of parameter estimates. The standard errors of the three parameter estimates K, M and β were 2·73, 2·41 and 0·23, respectively. This model accounted for ≈95·9% of the total variation. The optimum temperature estimated from Eqn 5 was 21·7°C. The minimum length (L) of wetness duration was highly correlated with M (r = 0·97) estimated using Eqn 3 and β (r = −0·92) estimated using Eqn 2. Regression analysis showed that L was related to M by the equation L = 2·06M with the constant term constrained to 0 (the standard error of the parameter estimate was 0·09). Thus the following equation described the relationship of L with temperature: L = 2·06M = 36·05 − 21·73T + 1·69T 3
(5)
where M was substituted by Eqn 3. Figure 3 shows the observed values together with the predictions given by both
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Figure 1 In vitro germination of Venturia nashicola conidia in free water over time at (a) 5; (b) 10; (c) 15; (d) 20; (e) 25; (f) 30°C. Symbols represent the observed germination of replicate experiments (three replicates for 10, 15, 20 and 25°C; five for 5 and 30°C); solid line = fitted logistic model with parameter values given in Table 2.
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Figure 3 Effects of temperatures on minimum length of wetness period (h) required for infection of detached leaves by conidia of Venturia nashicola. The observed minimum time for each temperature is shown as a triangle. Solid and dashed lines = fitted ‘direct’ (Eqn 4) and ‘indirect’ (Eqn 5) infection models, respectively.
Figure 2 Estimates of (a) rate parameter β; (b) time elapsed until 50% of maximum germination (M ) for logistic models describing temporal dynamics of in vitro germination of Venturia nashicola conidia in relation to temperature. Parameter estimates are also given in Table 2. Solid lines = fitted polynomial models for β (Eqn 2) and M (Eqn 3), respectively.
Eqn 4 and Eqn 5. These two models gave very similar predictions, except at low (27°C) temperatures.
Discussion The present studies show that conidia of V. nashicola can germinate over temperatures ranging from 5 to 30°C, with
an optimum at ≈21°C. Conidial germination needs nearsaturation humidity (RH > 97%). Percentage germination 24 h after inoculation was much lower in dry (no free water) conditions, or at extreme temperatures such as 5 and 30°C, than under other conditions. The low percentage germination observed may have been partially caused by greater conidial mortality in these conditions. A requirement for near-saturation humidity for germination was also reported by Luo (1983). In field conditions when RH is as high as 97%, it is likely that dew is present on leaf surfaces. In addition, commercial humidity sensors usually have an accuracy of ±3%. Therefore, for practical use of disease forecasting models, free water is essential for infection by conidia. For this reason, further germination and infection investigations were conducted under wet conditions only, despite significant interaction effects between temperature and RH on germination of conidia. The temporal dynamics of conidial germination under wet conditions (free water) were described well by logistic models for each individual temperature. The two parameters rate and time to 50% maximum germination were well related to temperature.
Table 3 Total number of Venturia nashicola conidia that infected detached leaves of cv. Ya-pear for each combination of wetness duration and temperature for 160 microscopic view fields at ×200 magnification over two replicate experiments. A conidium was considered to have infected a leaf if mycelium had developed under an appressorium
Temperature (°C) 10 14 18 22 26
Wet period (h) 6
7
8
9
10
11
12
13
14
15
16
17
18
20
– – 0 0 0
– –
–
– 0 34 19 20
–˙ 14 9 14 13
–
0 22 23 10
0 7 18 17 33
0 14 17 7 36
0 6 – – –
0 23 – – –
11 – – – –
32 – – – –
10 – – – –
18 – – – –
0 13 0
–, No observations made at this time.
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In this study it proved very difficult to obtain high quality data on the detailed temporal dynamics of infection of detached leaves by conidia. Instead, the observed data (Table 3) were used to obtain the minimum length of wetness duration required for infection of detached leaves by conidia. At 22°C, conidia took only 7 h to infect detached leaves. For the five temperatures tested, the minimum length of wetness duration for infection was just over twice that for 50% maximum germination estimated using the derived model (Eqn 3). Based on this constant ratio between durations required for 50% maximum germination and minimum infection, an ‘indirect’ model (Eqn 5) was developed relating the minimum length of wetness duration for infection to temperature. In addition a ‘direct’ model, relating this minimum length to temperature, was derived from the observed infection data without using the germination model. The indirect model was derived because the range of temperatures used to develop germination models was greater than that in the infection study. There were very few differences between the indirect and direct models in the temperature range 7 – 27°C, which covers the normal range (15– 25°C) of temperature in the early growing season in northern China. For general epidemiological characteristics, pear scab caused by V. nashicola is the same as pear scab caused by V. pirina or apple scab caused by V. inaequalis. The present models for the minimum wetness duration required for infection by conidia differ considerably from the model for V. pirina (Villalta et al., 2000). First, the model for V. pirina is exponential for temperatures in the range 4– 25°C. Second, the minimum time required by V. pirina is about 2 h longer than that required by V. nashicola for temperatures in the range 20 – 25°C. The models for V. nashicola in the current study are similar to the Africa /c model for temperatures
