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Maturation, Ascospores Discharge Pattern and Relevance of Mills Criteria for Predicting Apple Scab Infection Period in India



K.P. Singh, J. Kumar, A. Singh, R.K. Prasad, R.P. Singh and D. Prasad
 
ABSTRACT

Background: Apple scab, caused by Venturia inaequalis (Cke.) Wint. (anamorph Spilocaea pomi Fr.) is considered to be one of the most important fungal diseases of apple. In Uttarakhand Himalayas, the yield losses during scab epidemic years in 1996 and 2008 went up to 70%. Methodology: Random surveys were conducted during 1995-2014 from 3rd week of August until last week of September in apple orchards planted mostly to cv., Delicious. An apple scab forecasting and monitoring systems (μMETOS, Austria) installed at overwintering sites at Bhatwari fruit belt, Purola-Naugao, Tuni-Chakarata and Auli-Joshimath fruit belts recorded weather parameters including temperature, which were used to calculate degree-day accumulations. Results: The leaves falling early in the season appeared not to contribute much to the build-up of primary inoculum for the ensuing apple season. Leaves falling late had more scab lesions per leaf and posed a greater risk of increasing primary inoculums. The pseudothecia formation took 32-48 days more after sexual reproduction at temperature around 8-10̊C and this process was completed between February and March every year. The maximum ascospore productivity was recorded at fruit development stage of apple with ascospore productivity of 1,23,000 mL–1, which declined gradually. The ascospore emission period was 64-78 days and mean number of cumulative degree-days for 50 and 95% spore release were 456 and 960, respectively. Mills infection periods ranging from 19-47 were recorded from April-September every year that varied depending upon the weather conditions. The results revealed 2 day (light infection), 1 day (moderate infection) and 1 day (severe infection) delay in symptom expression under orchard conditions. Wetting requirement for infection to occur was also found to be more. More wetting of the leaves was required and symptoms were expressed within 7-9 days at higher temperature. Conclusion: The results of this study provide evidence that the use of average daily temperatures in spring to developed forecasting models based on degree-day accumulation could be used to predict the beginning and the end of the ascospore discharge during the apple growing season. The reduction of primary inoculum sources could have a decisive role in the management of apple scab.

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K.P. Singh, J. Kumar, A. Singh, R.K. Prasad, R.P. Singh and D. Prasad, 2016. Maturation, Ascospores Discharge Pattern and Relevance of Mills Criteria for Predicting Apple Scab Infection Period in India. Plant Pathology Journal, 15: 108-123.

DOI: 10.3923/ppj.2016.108.123

URL: https://scialert.net/abstract/?doi=ppj.2016.108.123
 
Received: January 09, 2016; Accepted: April 26, 2016; Published: June 15, 2016

INTRODUCTION

Apple (Malus domestica Borkh.) plays a major role in horticultural development and improving economic status of the growers in Uttarakhand Himalayas of India. The production of apples, however is faced with a number of challenges among which diseases are one of the important constraints facing apple farmers. Apple scab and powdery mildew are identified as the most important diseases of apples in the country with the potential to significant affect the temperate fruit agro enterprise1-4. Apple scab caused by Venturia inaequalis (Cooke) Winter (anamorph: Spilocaea pomi Fr.) is however, considered to be the most destructive disease. The disease had sporadic occurrence in the region until the year 1987 and by the year 1996 it could be recorded in about 10,000 ha approximately one fifth of the total area under apple in the districts Uttarkashi, Dehradun, Gwaldam and Tehri in Uttarakhand state. Yield losses during epidemic years in 1996 and 1997 in the region reached5 70%. In Bhatwari fruit belt alone scab has made the disease rendered nearly 23% of the crop unfit for either market consumption or processing. Infected apples were purchased by the government and dumped6,7.

Ascospores of V. inaequalis discharged from pseudothecia from over-wintered infected apple leaves are considered to be the sole constituent of the primary inoculum8-10,11. The time of leaf fall determines the number of pseudothecia that form per unit leaf area and the rate at which they develop12-15. Numerous studies have been conducted in different regions of the world to investigate maturation and discharge of ascospores14-21.

Ascospores discharge is dependent on various environmental factors, which ultimately affect quantitative production of ascospore inoculum in the spring. The ascospores are carried by the wind to susceptible young terminal leaves and fruits, where they cause primary infection leading to lesion development and production of conidia. The conidia are responsible for the secondary infections and epidemic build-up. During recent years, warning systems for plant protection have gained importance, especially in the context of “integrated plant protection22”. At present scab warning services have been most frequently used in Western European countries. In India, the scab warning services based on electronic scab warning systems, though in vogue in the states of Jammu Kashmir and Himanchal Pradesh have been organized lately in the major apple growing regions in Uttarakhand Himalayas15. Several studies have reported the use of sum of average daily temperatures in spring to develop forecasting models, which predict the beginning of primary apple scab inoculum release8,21,23-26. Most of the models are based on degree-day accumulation and could be used to predict the beginning and the end of the ascospore discharge during the apple growing season. Gadoury and MacHardy12 used data from laboratory and field studies in New Hampshire, USA to develop a model to estimate the cumulative percentage of matured ascospores and reported 95% spore maturation after 477 degree-days. The predictive models differ not in the shape of the curve describing maturation of a pseudothecial population, i.e., a sigmoid curve but in the number of degree-days required to reach a particular percentage of maturation. In South Africa, 50 and 95% ascospore discharge occurred when the degree-days cumulation (base = 0°C) from the date of first ascospore discharge is c. 500 and 900, respectively which is twice the number of degree-days required to reach the same levels of pseudothecial maturation with the model developed in New Hampshire. In France, the ascospore maturation also required a greater accumulation of degree-days to reach 50 and 95%, i.e., c. 325-400 and c. 500-775 degree-days, respectively, compared with the New Hampshire model. In Norway, 50 and 95% ascospores were mature in an orchard at 346 and 676 degree-day accumulations27. Thus, ascospore maturation and trapping cannot be the same with specific model predictions, since it is dependent on the occurrence of rain.

Mills28 considered (in the Mills table) three risk levels of apple scab infection: Light, moderate and severe in an orchard harbouring abundance of inoculum of V. inaequalis. The light level of infection identified the minimum hours of leaf wetness required for infection and the moderate and severe curves identified additional hours the leaves must remain wet for noticeable increase in scab incidence. Schwabe et al.29 developed minimum infection indices for light (<10%), moderate (10-40%) and heavy (>40%) scab incidence in which infection accounted for the percentage of leaves with scab lesions. Apple growers use Mills-based predictive systems to determine the risks of primary infection by ascospores30. Mills infection criterion requires a minimum leaf wetness of 9 h for ascospore infection and 5.9 h for conidial infection so as to produce symptoms in 9 days at the temperature range of 17.2-23.8°C31. Mills infection criteria for predicting primary infection has been widely adopted with modifications to suit local conditions15,30-35. However, several orchard studies15,31,35-37 have provided data that do not agree with Mills table. MacHardy and Gadoury35 suggested several changes to the Mills table and developed a new equation, based on the published data to describe the minimum infection time for conidia. This revision was further modified by Stensvand et al.36 described a shorter minimum infection time for ascospores and conidia at or below 8°C and speculated that both ascospores and conidia were often present simultaneously during the season of ascospore production and the required minimum infection time was similar for both the spore types. Thakur and Khosla31 studied the length of leaf wetness required for the majority of infection periods (Light, moderate and severe) and reported 1-3 days delay for symptoms development under Himachal Predesh conditions.

Weather conditions especially rainfall, duration of leaf wetness and temperature vary at different altitudes in the Indian Himalayan region where apple is cultivated. Therefore, understanding the timing and intensity of environmental conditions that trigger ascospore discharge may provide useful information to growers who use fungicides to protect their trees from new infection. Thus, the objectives of this study were to identify the pattern of primary inoculum, relation of degree-day accumulation to predict the beginning and the end of the ascospore maturation and ascertain the relevance of Mills infection period for predicting infection periods of V. inaequalis in farmers orchards at varied apple growing regions of the central Himalayas of India.

MATERIALS AND METHODS

Prevalence and severity of apple scab: Random surveys were conducted from the 3rd week of August until the last week of September to record scab incidence, ascospore maturation and weather data from apple orchards planted mostly to cultivar Red Delicious in Bhatwari fruit belt (1987-2014), Purola-Naugaon, Tuni-Chakrata, T al-Talwari, Bharsar, Auli-Joshimath, Munsiyari, Lohaghat and Almora-Muktheshwer fruit belts in Uttarakhand Himalayas in India from 1993-2007. Leaf wetness and temperature favoring scab infection varied from season to season from area to area and in some cases from orchard to orchard in the same area. In the study, the orchard was the sampling unit for measuring scab incidence with incidence expressed as the percentage of trees having scab infection. Severity was expressed as the percentage of leaf surface covered with scab lesions or the percentage of scabbed apples. Random surveys revealed an increasing trend in the prevalence and severity of disease in all such areas.

Time of leaf fall and decomposition: Spur and terminal leaves were monitored for scab development on unsprayed Red Delicious apple trees in commercial orchards at Bhatwari fruit belt, Purola-Naugaon, Tuni-Chakrata, Auli-Joshimath and Tal-Talwari fruit belts at 15 days interval from July until defoliation reached 100% in November. At each date, scabbed leaves were collected from the basins of the trees and packed in nylon mesh bags for overwintering on the orchard floor. From each collection, three samples each of 20 leaves were dried at 60°C for 24 h and weighed. Leaf area in each collection was measured using a leaf area meter (Leaf area meter, CID, USA). The relationship between percent defoliation and date of collection of fallen leaves was determined.

Pseudothecial maturity and ascospore productivity: Scabbed apple leaves from unsprayed orchards of Red Delicious cultivars were collected periodically from late dormancy to fruit development stages during 1990-2012. Leaves bearing pseudothecia were dipped in luke warm water (40°C) for 5 min to make the leaves pliable. Ten discs each of 1 cm diameter were cut using a cork borer from different leaves from each sample and separately boiled in lactophenol for 30 min in order to clear the tissue. Subsequently, each disc was examined under a stereo microscope and the numbers of pseudothecia were counted. Maturity of pseudothecia was assessed under a microscope using the method of Palmiter37 and Szkolnik38. The pseudothecia and ascospores were categorized on a scale from 1-5 where (1) No spores evident, (2) Colourless spores, (3) 10% coloured spores, (4) 25% coloured spores and (5) 50% spores coloured or discharged ascospores.

Patterns of pseudothecial maturity at different sites, Bhatwari fruit belt (2550 m a.s.l.), Purola-Naugao (2000 m a.s.l.), Tuni-Chakarata (2200 m a.s.l.), Tal-Talwari (1960 m a.s.l.) and Auli-Joshimath (2400 m a.s.l.) were similarly monitored on different dates. Ascospores were caught on glass slides smeared with glycerin and positioned 2 cm above scabbed leaves on the ground. Ten random observations from each slide were counted each time from each place. Spore count was made under normal light of Vanox Olympus research microscope.

Scab affected leaves of different cultivars i.e., Red, Royal, Golden Delicious, Rich-a-Red, Top Red, Red Spur Delicious, Starkrimson, Williams, Jonathan and Tydeman’s were collected from orchards prior to onset of leaf fall in autumn. Scab affected leaves (1 m2 leaf area) of each cultivars were placed in nylon mesh bags and these bags were overwintared on the orchard floor. Ten leaves of each cultivar were randomly taken out from bag and observed at intervals for the buildup of pseudothecial initials, pseudothecia with pseudoparaphyses and mature asci. Ascospore productivity which is the number of ascospores produced per centimeter square of dead leaf area was assessed by the method of Gupta and Lele39.

Degree-days: An apple scab forecasting and monitoring systems (μMETOS, Austria) installed at overwintering sites at Bhatwari fruit belt, Purola-Naugao, Tuni-Chakarata and Auli-Joshimath fruit belts recorded weather parameters including temperature, which were used to calculate degree-day accumulations. Cumulative degree-days were computed from daily maximum and minimum temperature values and base temperature of 0°C was used for all degree-day calculations. Degree-day accumulations were started at the date when the first ascospores were discharged. Timing of ascospore discharge was estimated up to 95% ascospore release and was related to cumulative degree-days at Harsil, Dharali, Jhalla and Sukhi villages in Bhatwari fruit belt. Degree-days and ascospore maturation were related using standard regression analysis. The ascospore discharge data of Bhatwari fruit belt was accumulated over 20 years and transformed using logit and probit transformations and plotted against the accumulated degree-days.

Mills validation: The relevance of Mills infection period was studied on new unfolded apple leaves after occurrence of infection periods and observed the infection according to the days given in the Mills infection table showing their relevance to the build up of scab spots on apple leaves. Weather data was recorded using microprocessor-based orchard environment monitor, RSS 412 apple scab predictor and μMETOS scab warning device. The data provided hourly records of precipitation, temperature, relative humidity and leaf wetness. The predicted infection periods were observed each year periodically from the green tip stage to the fruit development stage in an unsprayed orchard. The youngest leaves of 30 randomly selected terminals were tagged for observation after the predicted infection period on each rainy day. The total infection periods were observed in each year periodically from March till August at the Bhatwari fruit belt. Data accumulated over 20 years were analyzed for validation of Mills criteria to establish its relevance in rescheduling fungicide applications under monitored spray programme.

RESULTS

Status of scab: The prefectures with the greatest area under apple production are Uttarkashi, Chamoli, Pithoragarh, Almora, Dehradun, Pauri, Tehri, Nainital and Bageshwar (Fig. 1). Random surveys revealed an increasing trend with time (fruit development stage) in the prevalence and severity of disease in all these areas. The average rainfall in this region was 1238.7 mm and the relative humidity ranged between 70 and 80% during the apple season.

Fig. 1: Major apple-growing prefectures in Uttarakhand Himalayas ranked according to production

Fig. 2: Scab incidence in leaves and fruits of apple in Bhatwari fruit belt in Uttarakhand Himalayas over years

Table 1: Progress of scab in commercial cultivars of apple in Bhatwari fruit belt
*Mean value of observation on 100 leaves and 30 fruits per replication during 2004-2010

Portions of scabbed area (scab disease incidence) in the districts Dehradun, Uttarkashi, Chamoli and Tehri were 28.6, 54.8, 10.7 and 5.9%, respectively. In Chakrata fruit belt in district Dehradun, 5-75% trees were found affected though disease incidence remained low (5-30%) compared with Uttarkashi (30-95%). In Chamoli district, maximum disease incidence (21%) was recorded in tall village in Gwaldom fruit belt though in general, the disease intensity remained low (1-4%). The Chamba-Mussoorie, Almora, Pauri, Tehri, Nainital and Bageshwar fruit belts were apparently free of scab. A scab epidemic was observed in the Bhatwari fruit belt of Uttarkashi district during 1996, 2008 and 2013 with severe infection on leaves (30-86%) and fruits (70-95%). The most severe epidemic was observed in 2013 with 100% orchards and trees infected (Fig. 2, Table 1).

Time of leaf fall and decomposition: In Bhatwari fruit belt, defoliation in apple trees started around mid August and continued for nearly 65 days. Maximum leaf fall (82%), however was observed during the period 15 October to 15 November, after which trees were completely defoliated (Fig. 3). A linear relationship was observed between leaf defoliation (y = 3.929+2.716x, R2 = 0.993), dry weight (y = 47.571+40.393x, R2 = 0.963), leaf area (y = 3.928+2.716x, R2 = 932) and date of collection of fallen leaves. In Purola-Naugaon, Tuni-Chakarata and Tal-Talwari fruit belts, defoliation started by 15 September and continued up to 15 November. Maximum leaf fall was observed during October. A liner relationship was observed between leaf fall (y = 1.596+2.359x, R2 = 0.2771), dry weight (y = 18.964+37.702x, R2 = 0.951), leaf area (y = 1.7164+2.6111x, R2 = 0.9651) and date of collection of fallen leaves and could be used to forecast the leaf fall period in the orchards.

Pseudothecial maturity and ascospore productivity: Several factors affected the production of saprophytic stage of Venturia inaequalis.

Fig. 3: Leaf fall and decomposition of leaves in apple orchards of Uttarakhand Himalayas fruit belts

Fig. 4: Pseudothecial development stage of Venturia inaequalis at five location of Bhatwari fruit belt in Uttarakhand Himalayas

Development of pseudothecia and their maturation at the five sites in Bhatwari fruit belt is illustrated in Fig. 4, which depicts the proportion of pseudothecia at each development stage from October-June. While studying the decomposition of scabbed leaves fallen on the orchard floor during June-November, it was observed that leaves fallen during June-September were completely decomposed while leaves of subsequent months, when the actual leaf fall does occur and decomposed with efficiency in a decreasing order. Pseudothecia formation was initiated after several weeks of leaf fall and varied from site to site. Pseudothecia development started from November and December and progressed steadily when moisture and temperature conditions were favorable. The pseudothecia continued to develop asci until the end of February-April and ascospores discharged between May-June. In other districts of Uttarakhand i.e., Tehri, Dehradun, Pauri, Nanital, Pithaugarh and Almora pseudothecia were always found to have developed only up to the late filamentous stage during January to early March and did not develop any further. In fact at most of the sites, there was no development beyond the late filamentous stage until last week of March. Further development did not occur until after an extended period during which the pseudothecia grew in size and matured.

A relationship was observed between time of collection of fallen leaves (15 and 30 October, 15 and 30 November), leaf area, leaf fall, dry weight and production of pseudothecial (per 1 cm leaf disc) productivity (Fig. 5).

Fig. 5: Relationship between time of collections of fallen leaves, leaf area, leaf fall, dry weight and production of pseudothecial productivity

Fig. 6: Average number of mature pseudothecia and ascospore productivity in different fruit belts of Uttarakhand Himalayas

Collections made on 15 November showed maximum production of pseudothecia per centimeter leaf disc (146.4) and a linear relationship between leaf area, leaf fall, dry weight and pseudothecial production (y = -50.05+65.175x, R2 = 0.4204) from all collection sites was observed. Dry weight (316 mg per leaf) and leaf area (15.21 cm2 per leaf) were maximum in samples collected on 30 November, but the pseudothecial productivity was less (132.25 cm–1 leaf disc). Average number of mature pseudothecia and ascospores discharged per gram leaves were compared for several years at different locations in Uttarakhand hills (Fig. 6). Formation of pseudothecia and ascospore discharge were associated mainly with late autumn (October) infection. Maximum pseudothecia and ascospore production were observed in the 1996 and 2008 and least in 2000-2003.

The average progression of pseudothecial development could be depicted as proportion of pseudothecia matured in comparison to phenological apple development. Pseudothecia never matured before full bloom stage of trees. Pseudothecia continued to develop to the maturity stage 4 and 5 during petal fall to fruit development stages during second fortnight of May and 1st week of July. The seasonal variation of ascospore discharge at Uttarakhand is given in Fig. 7. Only 5-28% of total discharge was observed during most of the meteorological weeks, while the week 21 (Auli) and 23 had 37 and 68% of ascospore discharge, respectively. Maximum ascospores productivity was recorded at fruit development stage (pea size to walnut size) of apple with ascospore productivity of 1,29,000 mL–1 and thereafter declined gradually. This indicated that primary infection (ascosporic infection), which started in the Bhatwari fruit belt after May 2nd week continued till July 1st week. The secondary scab infection started from 2nd week of June and thus over lapped with the primary infection period. During 1998 and 2004, pseudothecial development and ascospore maturity were comparatively delayed, which was due to differences in prevailing temperature and leaf moisture at different phenological stages of apple. In most of the years, a relationship between ascospore productivity and primary infection of scab was found to be much higher (>90%).

Figure 8 shows pseudothecial maturation at Harsil, Gwaldam, Purola and Joshimath sites from full bloom to the fruit development stage (2nd week of March to last week of June).

Fig. 7:Seasonal variation of ascospore discharge at Uttarakhand Himalayas

Overall a pattern of pseudothecia maturation was observed from full bloom to petal fall stage (1st-3rd week of May) in Bhatwari fruit belt (Harsil) and Auli-Joshimath fruit belts at green tip to petal fall stage (2nd to last week of March) at Purola-Naugao and Tuni-Chakarata fruit belts and at fruit set (1 cm) stage (2nd week of May) at Tal-Talwari fruit belt. By the time trees attained fruit development stage (4 cm) at Bhatwari fruit belt, Purola-Naugao, Tuni-Chakarata, Tal-Talwari and Auli-Joshimath fruit belts, the pseudothecia had exhausted ascospores to the extent of 100, 88, 69 and 73%, respectively. The difference in the pseudothecial maturity and emission period in relation to tree phenophase could be due to weather conditions prevalent during pseudothecia development and maturity.

As is evident from Fig. 9, apple cultivars did influence the production of pseudothecial initials, pseudothecia with pseudoparaphyses and the mature asci, thereby resulting in maximum production on the leaves of Royal and Red Delicious. The minimum production of pseudothecial initials and pseudothecia with pseudoparaphyses occurred on the leaves of Tydemand’s Worcester, which otherwise had provided the maximum number of mature ascospores.

Fig. 8: Ascospore maturity at four different places of Uttarakhand Himalayas

Fig. 9: Effect of apple cultivars on the pseudothecial maturity and ascospore dose of V. inaequalis

Fig. 10: Ascospore discharged from over wintered scabbed leaves at Bhatwari fruit belt in Uttarakhand hills

Table 2: Degree-day for primary infection of scab in different apple growing belts of Uttarakhand hills

However, the maximum proportion of asci with mature ascospores were produced on leaves of Red, Royal and Golden Delicious in the Bhatwari fruit belt. The remaining cultivars were found to be intermediate in production of pseudothecial initials, pseudothecia with pseudoparaphyses and mature asci. These observations thus revealed the influence of host cultivars on the perennation of V. inaequalis which not only affected the pseudothecial density but also resulted in variable patterns of potential ascospore dose in Bhatwari fruit belt.

Predictive maturation model for ascospore discharge: A sigmoid curve was obtained when accumulated degree-days were plotted against cumulative percent discharged ascospores. The curve was considered to occur in three identifiable phases i.e., lag phase, accelerated phase and final phase and was used in the apple scab management program of Uttarakhand (Fig. 10). The slow buildup of matured ascospores during lag phase is relatively easy to control with fungicides (Post infection strategy). At the time of accelerated phase, the crop is potentially at high risk, if proper control is not exercised. Protectant or post infection strategies could be employed but a protectant schedule is usually preferred, especially if scab could not be managed well during the previous year. The final phase is significant as it identifies the end of the secondary infection season with emphasis on selecting the scheduling fungicides to control other diseases provided the scab keeps on deteriorating at the last phase.

The mean number of cumulative degree-days for 50 and 75% ascospore discharge were between 279-900 and 412-1080 DD, respectively (Table 2). Ascospore maturation data of 20 consecutive years was pooled and plotted against degree celsius day accumulation from the date of 1st ascospore discharge in the Bhatwari fruit belt region and is presented in Fig. 10.

Fig. 11: Ascospore discharged from over wintered scabbed leaves at different place of Uttarakhand Himalayas

The dry periods in spring during 1999-2003 in Uttarakhand hills might have retarded ascospore maturation and such conditions might have coincided with a delay in ascospore maturation in the region. A positive correlation was observed between the ascospore maturity and temperature during all the study years except for 1999 and 2000. In Uttarakhand hills at high altitudes (Gangothri fruit valley and Auli), winter was generally cold until late April and the apple cultivars were in the green tip stage until late March. At other places the same phenological stages were found one month earlier. Therefore, the seasonal variation played an important role in the maturation of ascospore in Uttarakhand Himalayas.

Ascospores were recorded at 8 places viz., Harsil, Sukhi, Auli, Joshimath, Koti-Kanasar, Sauri, Tal-Talwari and Gwaldam. The higher correlation was obtained when probit and logit transformations from each place were subjected to linear regression. A significant relationship between degree-days and probit/logit is the first indication for the prediction of ascospore maturity based on daily temperature. It was ended up with the same linear relationship with minute differences in both the cases i.e., Y = 4.247+1.22x, R2 = 0.856 (probit) and Y = -0.225+0.003x, R2 = 0.859 (logit). Both the relationshpis were precisely the same so, it was preferably used probit transformation for predicting ascospore maturation against degree-days accumulation because a significant linear relationship was observed and it allowed the prediction of primary inoculum maturity based on daily temperature.

Quadratic and linear regression models of 8 different places viz., Harsil, Sukhi, Auli, Joshimath, Syori, Koti, Talwadi and Gwaldam (R2 = 0.9276, 0.9278) fit the data better and coefficients for terms in both equations were significant (Fig. 11). Linear and quadratic regression indicated a curvilinear relationship between cumulative percent ascospore matured and degree days. Regression results were similar using either cumulative percent ascospore matured and degree-days non adjusted or adjusted for disease development; predictions of disease severity with these two quadratic regression equations differed by only about 1%. Results of both analysis were similar, indicating that it might be possible to eliminate multiplication of pathogen in further studies on temperature of scab, especially when less favourable temperature condition is present for disease development.

Validation of Mills infection periods: On examination of 20 years data of primary infection periods from Bhatwari fruit belt, differences in respect to ascospore infection when compared with a Mills table were observed. The environmental conditions varied from location to location and Mill’s infection periods ranging from 19-47 recorded from April-September every year revealed that number of infection periods varied from year to year depending upon the weather conditions (Fig. 12).

Fig. 12: Occurrence of apple scab infection periods in Bhatwari fruit belt of Uttarakhand Himalayas

The maximum numbers of infection periods were recorded in Bhatwari fruit belt, which resulted in the moderate to severe build up of scab during the ensuing periods. As indicated by the infection periods, weather conditions were very favorable for the development of scab epidemic in Bhatwari fruit belt. For scab infection to occur, the spore on the leaf surface must remain wet long enough for it to germinate and grow into the leaf. The length of time required is related to the temperature. Young tagged leaves of 30 terminals were observed for the development of new scab lesions after the stipulated time interval. The data thus obtained were compared with the criteria established in Mills table. First infection period was encountered on 14 May and correspondingly the apple scab lesions could be seen in the orchard after 15 days which is differ approximate time of symptom appearance as per the Mills table. Similarly, other infection periods falling in May, June and July had relevance to the development of scab after indicated periods placed in Table 2. The revised Mills table indicates the minimum number of hours of continuous wetting periods required for primary infection of apple leaves by ascospores of Venturia inaequalis.

It was defined minimum infection time as the minimum time required for successful infection of any quality of tissue on trees. This observation indicated that at 10°C, according to Mills table, the time needed for symptom appearance was 16 days. Figure 13 shows 4-8 light infection periods occurring each year during the months of March, April and May which could initiate primary infection. The time required for symptom expression was 9-14 days under prevailing temperature conditions. The infection time was more than the predicted (1-4 days) as per Mills table. Six to 10 moderate infection periods were recorded in each month during 1990-2012 and almost all indicated delay (1-3 days) in the symptom expression under orchard conditions (Fig. 13). The third criteria as described by Mills were severe infection period. Two to 13 infection periods were observed in most of the months at an average temperature (11.4-15.2°C) and leaf wetness (23.4-27.2 h) period and indicated 1-2 days delay in symptom expression (Fig. 13). For validation of criteria for scab prediction, the collected data was analysed to establish its relevance in rescheduling fungicide application under monitored spray programme in the orchards. It was defined that this minimum infection time as the minimum time required for successful infection of any quality of tissue on trees. This observation revealed 2 day (light infection), 1 day (moderate infection) and 1 day (severe infection) delay in symptom expression under orchard conditions. The regression analysis was used to describe relationship between Mills infection criteria and the data on light, moderate and severe infection period for symptom appearance (Fig. 13) in orchards. In all the cases, the total variation was high in low, moderate and severe infection curves. After having studied different inoculum levels under Garhwal Himalayan conditions, it could be inferred that reduced spray program could be used after petal fall for the management of scab.

Fig. 13(a-c): Primary scab development on terminal shoots in under orchard conditions, (a) Light infection period, (b) Moderate infection period and (c) Severe infection period (dotted line for Mills table and black line for revised)

DISCUSSION

The growth dynamics of V. inaequalis is governed by the interaction of host, disease and inoculum potential each of which could be affected by environmentally driven variables, such as leaf wetness and temperature. These factors are known to play a decisive role in the perennation of apple scab pathogen in the perfect stage in fallen leaves and subsequently in the events leading to initiation and build up of the disease. In absence of the knowledge about the role of these factors on perennation, it had not been possible to make rational spray decisions. Much of the study on the epidemiological aspects of the disease has been else where in the world. As such, modified Mills curves have been developed in Australia, Belgium, France, Netherlands, South Africa and Switzerland. Whether similar situations exist in India is still not known. In India, the study on this aspect, however was confined to Himanchal Pradesh and laboratory conditions15,21,31,39 and there appeared a need to ascertain the known effects of weather on the perennation of scab fungus under field conditions. In the present study, scab disease incidence increased due to increase in rainfall, relative humidity and decrease in mean temperature. It could be said that the disease on fruits and leaves was favoured by high relative humidity, continuous intermittent drizzling and moderate temperature. However, Gupta and Lele39 and Tomerlin and Jones40 reported maximum disease severity in years of more than normal rainfall. Weather conditions favoured scab at all the places during all seasons though it varied from season to season. In 1996 growing season at Bhatwari fruit belt, severe scab occurred early in the season, although weather conditions at the time were not particularly favourable for scab. The main reason for the heavy infection appeared to be the high inoculum level from the previous season. Continuous wetting periods resulted in moderate to heavy infection in orchards with high infection potential during the summer period of the growing season.

Venturia inaequalis survives the winter mainly as pseudothecia on dead, scabbed apple leaves on the orchard flour. Conidia and mycelium of V. inaequalis do not survive freezing of colder climates26,41,42. Ascospores produced within pseudothecia on fallen infected leaves constitute the principal and often sole source of primary inoculum for apple scab7,43. The present findings reveal a significant relationship between the time of leaf fall and leaf decomposition, which plays an important role in maturation and discharge pattern of ascospores. The leaves which fell earliest in the season, showed maximum reduction in leaf weight and area. It could, therefore be concluded that earlier the leaf fall better the decomposition of apple leaf litter during the over wintering stage. The leaves falling early in the season (August and 1st week of September) would not contribute much to the build up of primary inoculum for the ensuing apple season. In the Uttarakhand Himalayas, leaves falling late are likely to have more scab lesions per leaf and pose a greater risk of increasing primary inoculums.

The present study revealed that temperature had a positive correlation with the maturity and subsequent liberation of ascospores. Seasonal variation in conditions affecting ascospore release and survival may alter the proportion of ascospores that are available for new infections. Variables that regulate ascospore availability include the number of ascospores produced in each pseudothecium, the rate of ascospore maturation and the timing and intensity of climatic conditions that trigger spore release. The peak discharge of ascospores was found at full bloom to petal fall stage of apple tree at most locations accept the Bhatwari and Auli-Joshimath fruit belt, which is in agreement with the finding of Singh and Kumar5. However, dry winter months delay the maturation of ascospores13. Based on these observations, the phenological data on pseudothecial development indicated that most of the pseudothecia were mature by the end of winter in under Uttarakhand Himalayas. Low temperature (>8°C) after leaf fall for 54-69 days initiates sexual reproduction from the first rainy day or under optimum moisture conditions. The pseudothecial formation takes 32-48 days more after sexual reproduction at temperature around 8-10°C and this process completes between January to February every year in Uttarakhand Himalayas. The observation revealed that 96% of pseudothecia were matured at green tip stage during 2nd week of March at Purola-Naugao, Tuni-Chakarata and Tal-Talwari fruit belts (1700-2000 m a.s.l.) while at Bhatwari and Auli-Joshimath fruit belts (2100-2600 m a.s.l.) the pseudothecia started becoming mature at late pink bud stage. The ascospore emission period was 64-78 days and mean numbers of cumulative degree days for 50 and 95% spore release from these observations were 456 and 960. Occurrence of leaf fall before October inhibited the pseudothecial formation in the usual dry spell during the following months up to December. The ascospore maturity was highest at fruit development stage (walnut size) in the last week of May to 1st week of June as an average 72% pseudothecia had coloured spores with 56% getting exhausted. The ascospore discharge season was over within 42 days at one place as against 64 days at another place, while in earlier years it was over within 56-78 days. Such trends thus reflected the prolonged dry weather conditions in winter and spring with meager snowfall not allowing normaldevelopment of pseudothecia during 1998-2004. Mills and La Plante44 indicated that 3-4 h of dry foliage will stop the infection process which, however, also depends on the humidity. Similar results have been reported on the length of the dry period of leaf wetness45.

The cultivar susceptibility studies showed that the maximum number of pseudothecia had discharged in the Bhatwari fruit belt (2600 m a.s.l.) as opposed to decreasing pseudothecia development from the leaves of all cultivars in the orchards situated at lower altitude (1960 m a.s.l.) i.e., Tuni-Chakarata and Tal-Talwari fruit belts. This was the effect of prevalent cooler temperatures and humid conditions, being much cooler and humid at Bhatwari fruit belt compared to Joshimath, Purola-Naugao, Koti-Kanasar and Gwaldam with respect to the development of pseudothecia on overwintered leaves. Percent pseudothecial maturation and ascospore productivity was much higher in fallen scabbed leaves on orchard floor in comparison to those leaves that were kept at a sunny site in Bhatwari fruit belt. This trend of production of pseudothecia reflected the role of humidity, which was constantly high and kept the leaves pliable in shady site of the orchard. Gupta and Raj13 showed that there was no influence of orchard height on the pseudothecial maturity and ascospore dispersal period. In this study the orchard heights represented three different weather conditions i.e., severe, moderate and mild winters. It was infact the outcome of weather conditions prevalent during overwintering period. For this reason, reduction of primary inoculum sources could have played a very important role in the improvement of effectiveness scab management of apple in Uttaranchal Himalayas.

In several countries, growers use Mills28 criteria as an advisory for curative spraying against apple scab, independent of the history of disease control26. Mills table for infection was developed after many years of trials and observations in the field. Some of the difference between our results and those of Mills and La plante44 for infection by ascospores could be due to temperature, duration of total wetness period containing an abundance of inoculum released and germination. The same was received due to relationship between temperature, duration of total wetness period containing an abundance of inoculums released and germination. The prevailing microclimatic conditions, topography and apple cultivars might be the possible reasons for the delay of ascospore release and symptom development in Uttarakhand Himalayas. The infection periods recorded with the help of apple scab predictor, varied from year to year (1990-2012) depending upon the weather conditions and there were 19-47 Mills infection periods from early May-September in Bhatwari fruit belt. In Uttarakhand, apple scab predictor and μMETOS were able to predict infection periods correctly as tagged leaves showed new scab lesion accordingly. Once infection is predicted and there is availability of mature ascospores, relevance of these infection periods as predicted by the apple scab predictor and other weather monitoring equipment, could be seen under orchard conditions to judge whether the Mills infection period table is valid under Indian conditions or not. May being the time of most critical phenological stages (bloom or near to petal fall) allowed primary infection beyond light intensity when the subsequent wet weather prevailed in this month. All the levels of infection periods i.e., of low, medium and heavy intensity were able to cause initiation and development of scab lesions at Bhatwari fruit belt. These observations further made it clear that at high temperature, wetting period of small duration could also cause infection thereby remitting in production of symptom within a short period of 7-9 days. The ascospore maturity started around 1st week of May and continued up to end of April at different locations in Bhatwari fruit belt. In other places of Uttarakhand hills, the ascospore maturity started around 1st week of March and continued up to mid May. In the month of July, a severe infection period was observed at Bhatwari fruit belt and correspondingly the apple scab lesions could be seen in the orchard after 9 days. Similarly, other infection periods of light and moderate intensity falling on 2nd and 3rd week of June, respectively had relevance to the development of scab after indicated period. The relationship of leaf wetness and temperature for ascospore release and infection are consistent with the finding of others11,31,32,34,36. MacHardy and Gadoury35 also suggested revision of Mill’s criteria for infection. For validation of criteria for scab prediction, the collected data was analysed to establish its relevance in rescheduling fungicide application under monitored spray programme in the orchards.

CONCLUSION

The results of this study provide evidence that the reduction of primary inoculum sources could have a decisive role in the management of apple scab. There is a risk of early scab epidemics initiated by over wintering inoculum in the orchards where there had been a high scab incidence in the previous seasons. As several factors are involved in calculating the pattern of distribution of ascospores, such as amount of overwintering inoculum, cultivars, size of tree, spacing, wind velocity, leaf fall, leaf wetness, temperature and rate of leaf decomposition results of the present study may not be applicable to all orchards of Indian Himalayas. Studying the development of scab fungus in relation to weather conditions, moisture either in the form of dew or rain had greater impact in initial development of pseudothecia within 30-40 days after leaf fall. Temperature and rainfall were also influence the maturation of ascospores. There were enough mature ascospores present in the overwintering leaves to allow for some ascospore discharge by the time of silver tip stage. Maximum ascospore productivity coincided with the full bloom to petal fall stage in Uttarakhand hills. Such routine observations on ascosporic phase in relation to tree phenology were valuable in deciding about the initiation of timely control programme in the orchard. Nevertheless, the relative significance of apple scab predictor was as effective as the Mills table in predicting scab infection periods under Uttarakhand Himalayan conditions. Studies conducted over 20 years revealed that in majority of the infection periods, time taken for symptoms to appear was 1-3 days more than studyed out by Mills. Since, the observations were based on the events taking place under the natural environmental conditions, there appears to be the need to carry out studies both under controlled and natural conditions to study out the requirement of exact hours of wetting at a particular temperature and the period required for incubation. Such observations are required to be taken in different apple growing areas of Himanchal Pradesh and Jammu Kashmir for a couple of years to suggest changes in the Mill’s criteria for predicting scab infection periods.

ACKNOWLEDGMENT

This study was carried out utilizing the grants received under NATP-CGP III of the ICAR. Facilities made available by the Dean, College of Forestry and Hill Agriculture, Hill Campus, Ranichauri are gratefully acknowledged.

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