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Pages: 15-25

Date of Publication: 22-Aug-2021

Effect of temperature and relative humidity on conidial germination of the causal agent of cucumber powdery mildew

Author: Nagah Milod, Gazala Saad, Hanan.A.Khalifa

Category: Health Science

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Abstract:

This study was conducted in the plant protection department, faculty of agriculture and botany department, faculty of art and Science in AL. Gubba. Omar AL-Mukhtar University during season 2019-2020. The effects of relative humidity (RH) and temperature on conidial Germination of E. cichoracearum were studied in controlled environments to define conditions that affect the disease development of cucumbers in AL.Gabel, AL-Akhder region. Libya. Gradients of RH (20–90%) at constant temperatures (20–30__degreesignC) were generated in single growth chambers to determine their effect on mildew development on mature cucumber plants, temperature from 5 to 35 are evaluated their effect of Temperatures of 30__degreesignC and above were deleterious for spore germination, germ tube elongation, and disease development Lesion growth and rate of disease progression were significantly higher at 20__degreesignC than at 25__degreesignC. Low RH levels (20–40%) reduced spore germination and lesion growth, accelerated host tissue death, and reduced disease progress. Intermediate RH levels (50–70%) increased spore germination and optimized disease development, provided temperatures were maintained within favorable limits. High RH levels (80–90%) were favorable for spore germination but continued exposure to these conditions led to a limited lesion growth and disease progress. Short daily periods (two or three daily exposures of at least 2 h) of high temperatures (35__degreesignC) suppressed disease development by 70–92%.

Keywords: Cucumber powdery mildew, conidial germination, environmental factors

Full Text:

Introduction

Basal Powdery mildew is a term derived from the general observation that plant parts affected by the disease usually appear as if they have been dusted with fine white powder, This fine white powder is, in reality, a mass of abounds out mycelia, conidiophores, and conidia of the fungus, which normally grows on the surface of the host. The causal fungi themselves are known to spend the whole of their life cycle as obligate parasites. Powdery mildew is common, widespread, and very present among crop plants and ornamentals. The total losses in plant growth and crop yield it causes each year on all crops probably surpass the losses caused by any other single type of plant disease [43]. At one time Erysiphe cichoracearum D.C. was believed to be the only causal organism of powdery mildew on Cucumber in Libyan [24, 26, 38]. Later, Sphaerotheca fuliginea (Schlecht) was recognized on the basis of conidial characters, as a causal organism of powdery mildew on cucumber which is widespread in Libyan country, and a similar finding had been reported earlier from Sudan [14, 36, 45]. Powdery mildew disease occurs most readily in dry, warm areas with cool nights. Germination begins when the conidia land on the leaf during warm, dry conditions [21]. After nightfall, germ-tube production occurs and the infection process proceeds as humidity level rises [21]. The effects of moisture on conidial germination and penetration, and powdery mildew disease in general, are controversial [5]. The disease can reduce the photosynthetic area of leaves, and in severe cases causes defoliation of plants, effects that are likely to reduce the yield and quality of fruit [8]. According to professional and scientific literature this fungus is very harmful because of its direct effect on plants nutrient uptake from leaf tissue and indirect harmful effect since its epiphytic mycelium reduces assimilation by covering the leaf surface [31]. During the vegetation period, the fungus forms several micro cycles of the conidial stage, so its infectious potential on the leaves increases exponentially in favorable conditions, new conidia are formed from a mature conidium in 3-4 days. Because of its harmful effects, this fungus was and still is the subject of interest and intensive study from various aspects. A less studied aspect is the part of its life cycle from the time of conidial maturity to hyphal penetration into host leaf tissue This period is important for further development of the fungus and thereby infection of the host plant During an interval of favorable conditions, which in nature do not last very long, conidia germinate and form germ tubes that infect the leaf tissue by developing secondary hyphae. In addition to external conditions, a critical factor in infection is the susceptible phenological stage, i.e. the presence of young leaves [12, 13, 19, 33, 48]. Different powdery mildew fungi are reusing different optimum conditions for disease development [39]. The environmental factor also influences germination, formation release and survival of spores as well as mycelium development [37]. The effects of various environmental factors on powdery mildews vary with the species studied and the conditions under which it is studied. This variation has led to considerable confusion as to the effect of the environment on the development of powdery mildews in general. The factors that will be treated are temperature, moisture. Although the powdery mildew initiates infection from ascospores, conidia, and overwintering mycelia in dormant buds the conidial stage is the most important spore form in a secondary spread. Most of the studies on environmental effects have been made with this spore form. The aim of this study was to evaluate the importance of basic environmental factors such as temperature and relative humidity, on the germination of cucumber powdery mildew conidia and, based on the obtained results, to clarify their roles and place in the epidemiology of this pathogen.

Materials and Methods

Source of inoculum

The inoculum was collected from diseased plants in the EL-Wasiata region and identified in the plant protection Lab.

Identification of the causal agents

Collected samples were tested in the plant pathology Lab. For the presence conidial stage, measurement of conidia thickness of mycelium and shape of germ tube [15].

Effect of temperature on conidia germination

Conidia of E. cichoracearum from a diseased leaf (Fig.1) were dusted onto glass microscope slides using a paintbrush. Two slides were suspended on a rubber bung sited over water in a sealed plastic container (10 x 50 cm depth). Individual containers were placed in incubating rooms at a range of temperatures from 5-35__degreesignC (Fig. 2), with five replicate containers for each temperature treatment. After 3 days incubation of conidia developed in full light and relative humidity was set at 90%., the percentage of conidial germination was determined using a microscope [7, 44].

Effect of relative humidity on conidial germination

Conidia of E. cichoracearum were dusted onto microscope slides which were suspended over the solutions in plastic containers (as above). The containers were then sealed and placed in an incubator (__degreesignC) to give the required relative humidities (NH4NO3 63%, NaCl 75%, KCl 86%, KNO3 94%; H2O 100%. Percentage germination was determined with a microscope after 3 days incubation (O’Brien 1948).

The conidial germination dynamics and germ tube lengths were determined microscopically after 4, 8, 12, and 24 hours after the onset of incubation, using the experiments were conducted in three replicates for each treatment. In each replicate, germ tube lengths of 30 conidia were measured to obtain their average lengths.

Growth and inoculation of host plants

Seeds of Cucumis sativus cv. Beit alpha and M2/E6811-42191 were grown in a mixture of Levington’s compost and sand (1:1) in growth room conditions (20 ---PlusMinusSymbol--- 2__degreesignC, 16 h irradiance, 80 µmol m?2 s Uniformly developed seedlings were transplanted into plastic pots (12 cm diameter) containing compost, and allowed to continue growth under similar conditions. When the first leaf was fully expanded, plants were placed in the base of a settling tower (diameter 60 cm; height 80 cm) and inoculated by exposing them to an even distribution of S. cichoracearum conidia from heavily at different degrees of temperature and relative humidity as mention above. Infected leaves of cucumber, which had been shaken 6 hours previously to remove older spores [3, 16]. Primary germ tubes emerged within 3–6 hours, first haustoria within 12 hours after inoculation, and a second germ tube developed during the following 6 hours Within 24 hours after inoculation, septate hyphae were initiated from the primary and secondary germ tubes, giving rise to surface mycelium from which conidiophores began to develop by 5 days after inoculation, with conidia densely covering the leaf surface by 7 days Small pieces of host leave having numerous ascospores of the powdery mildews were submerged in sterilized distilled water for varying periods and after drying were subjected to desiccation. These leaves were then transferred to filter paper which helped in mounting the ascospores on slides for subsequent observations.

Disease development on individual plants

Disease Assessment

Disease Incidence

Percentage of each foliar disease incidence was recorded as the number of diseased plants relative to the number of growing plants for each treatment, and then the average of disease incidence in each treatment was calculated.

Disease Severity

Percentage of each foliar disease severity was recorded as the following equation

Disease Severity % = __ampersandsignSigma; (a x b) / N x K x 100

Where: a = Number of infected leaves in each category.

b = Numerical value of each category.

N = Total number of examined leaves.

K = The highest degree of infection category.

N = Total examined leaves

Numerical value from 0 to 4 according to Cohen et al. (1991) was followed, whereas: 0 = No leaf lesions; 1 = 25% or less; 2 = 26-50 %; 3 = 51-75 %; and 4 = 76-100% infected area of plant leaf. At the end of growing season the accumulated yield was calculated for each particular treatment.

Disease assessment: Fourteen days after challenge inoculation, powdery mildew disease development-as affected by the different tested treatment-was evaluated by counting the number of mildew colonies on the leave’s surface with the naked eye.

Disease development was monitored on five individual cucumber plants at the lab of plant pathology. As the plants grew, each leaf was numbered and the date of emergence was recorded. Leaf length measurements (from leaf base along the central vein) were made at 3-4 day intervals (ref). Disease development was also monitored on individual leaves. These measurements were carried out over two growing seasons (2019\ 2020) and (2019/ 2018).

Data analysis

Data were subjected to statistical analysis using randomized complete block design (RCBD) and ANOVA was used to determine the relationship of powdery mildew with environmental conditions was determined by correlation regression analysis [42].

Results and Discussion

Identification of the causal agents

The causal agent identified according to microscopic examination to the conidial stage which appears chains of parallel spores on short branched conidiophores, absences of ferionic bodies in conidia germ tubes no branched, according to these data the causal agent identified as E.cichoracearum, and confided in plant pathology institute Elgiza-Egypt.

Effect of temperature on conidium germination

The greatest germination (up to 55.2%) was recorded at 25__degreesignC, although germination was generally low after 3 days of incubation (Fig. 1). Some conidia germinated at 20__degreesignC and30__degreesignC but no germination occurred at 5__degreesignC or 35__degreesignC The leaves with infection were submerged in sterilized distilled water and were incubated at 0__degreesignC, 5__degreesignC, and15__degreesignC. For varying periods and were afterward incubated at laboratory temperature (25__degreesignC, f2®C.). The results are presented in Tables (1). At all the temperatures, the best results were obtained in a material subjected to low temperatures hours and alternating with exposure to laboratory temperature (25__degreesignC).

Table 1: Average length of germ tubes at different temperatures after 24 hours incubation.

It is generally accepted that germination of conidia of E. cichoracearum occurs between 15__degreesignC and 30__degreesignC, and is greatest at 25__degreesignC according to [20, 32].

The highest average length of germ tubes was recorded at 25__degreesignC at all of the stated time intervals (Table1). At temperatures lower and higher than optimal, germ tubes growth was significantly lower, while at 5__degreesignC and at 35__degreesignC the conidia did not germinate to this environmental factor. Their fore temperature is not a restricting factor in the germination of conidia, as powdery mildew occurs in temperate zones with prevailing favorable temperature conditions for its occurrence and spreading. Our experiment observations indicate that the disease does not appear on cucumber

Until the air temperature emits above 20__degreesignC. The disease spreads rapidly during the hot period. The best conditions for disease development are 35__degreesignC and more than 70% relative humidity. According to [50] powdery mildew infects plants within a temperature range of 11__degreesignC-28__degreesignC [2] reported that powdery mildew is generally favored by relatively dry atmospheric conditions, moderate temperature, and plant growth.

This temperature range falls within the summer range in the AL. Gabel, AL. Akhder region, when powdery mildew starts to appear on Cucumber crops. These laboratory results may partially explain our field observations that powdery mildew symptoms appear first on leaves under dense canopies where the relative humidity is usually high. Development of mildew in relation to weather factors Weather conditions play an important role in the appearance and rapid build-up of especially the maximum temperature.

Effect of relative humidity on conidium germination Results in table 3 indicated that germination high was directly proportional with relative humidity, high conidium germination 28.4 occurred at 100% relative humidity.

Table 2: Saturated-salt solutions used and relative humidity as measured in chamber studies

Table 3: The percentage of germination of Cucumber powdery mildew conidia at different temperatures after culturing under 25__degreesignC after 24 hours incubation, according to the results of Duncan’s test (10).

Relative humidity expressed a very weak correlation with the development of powdery mildew of Cucumber The infection process of Erysiphe cichoracearum on host cucumber was studied in this paper. The appressoria of conidia formed at similar rates on cucumber leaves, indicating that no resistance was expressed during the pre-penetration stage of Erysiphe cichoracearum [23] reported that the effect of relative humidity on spore germination. Relative humidity as low as 3% showed 34% germination and the highest (92% germination was observed at 100 percent relative humidity). Conidia germinate best at a relative humidity of 97-100% [1], but not below, indicating that they require moist air to germinate. Butt (1978) who demonstrated that germination decreased or was delayed in the presence of free water our results is an agreed also with [18] who found that conidial germination of powdery mildew of cucumber was maximum at a temperature of 25__degreesignC, 100__degreesignC/ R.H These laboratory results may partially explain our field observations that powdery mildew symptoms appear first on leaves under dense canopies where the relative humidity is usually high development of mildew in relation to weather factors Weather conditions play an important role in the appearance and rapid build-up of especially the maximum temperature [2] reported that powdery mildew is generally favored by relatively dry atmospheric conditions, moderate temperature and plant growth [23] reported that the effect of relative humidity on spore germination. Relative humidity as low as 3% showed 34% germination and the highest 92% germination was observed at 100 percent relative humidity [27] found sunshine to be more important than R.H. for development of powdery mildew on mung bean in Madhya Pradesh Sharma [45] studied the development of powdery mildew and fund that early October sowing favors more disease development due to favorable weather conditions. Lack of information on the effect of humidity on the development of different stages of powdery mildew keeps us from knowing whether the phenomena described for E.cichrocerum on Cucumber apply to other powdery mildews as well with E. cichoracearum the colonization-sporulation and dispersal were favored by dryness while infection and survival phenomena were favored by high RH or even wetness. However, in all cases, these were preferences but not essential conditions, as mildew developed also under less favorable conditions. This situation was reflected in the relatively small differences between final levels of epidemics induced in growth chambers under various conditions of RH. The fact that these epidemics reached the highest level at low RH, suggested that under the temperature conditions of 25~ by day and 15~ by night, dryness slightly favored the epidemiological patterns Conidia germination was optimum at 18 and 20__degreesignC and with a relative humidity of 70 and 90%. The association between relative humidity and temperature varied at different temperatures, with a non-significant effect of RH

Effects of environmental factors on disease development.

There was a similar pattern of powdery mildew development on all Cucumber plants (Fig. 2). Symptoms did not appear on the first leaves after planting until 7-8 weeks after emergence, but appeared progressively earlier, relative to the age of the leaf, on the later formed leaves (2 weeks). Symptoms rarely developed on leaves that had not grown to their full expansion and were never recorded on rapidly growing leaves Growth and rate of disease progress increase in the maturation of ascospores were obtained experimentally when the duration was increased, time at 25 Small pieces of host leaves having numerous ascospores of the powdery mildews were submerged in sterilized distilled water for varying periods and after drying were subjected to desiccation at 25__degreesignC and 30__degreesignC. For periods in incubators. These leaves were then transferred to filter paper which helped in mounting the ascospores on slides for subsequent observations. The results are presented in table 2. The data in table 2 show that alternate wetting. and drying of the ascospores material gave the best results on the ascospore formation when subjected to 30__degreesignC but not at other temperatures [9] have reported similar results with Erysiphe graminis.

Maximum incidence of powdery mildew was observed at air temperature 34-35__degreesignC and 18-19__degreesignC, maximum and minimum temperature respectively (Fig.2 and Fig.3). At these temperatures incidence of PM continued to increase. Our experiment observations indicate that the disease does not appear on cucumber so the air temperature emits above 20__degreesignC. The disease spreads rapidly during the hot period. The best conditions for disease development are 35__degreesignC and more than 70% relative humidity. These results are in line with earlier reports on the effect of accurate bleaching on environmental factors [30]. These results and observations are in accord with those obtained by [17] with Erysiphe graminis var. hordei through the exposure of the material to 9__degreesignC alternating at 21*^C [29] while studying the role of weather factors for the development of powdery mildew on urd bean in the Rabi season found maximum temperature between 21.0-26.1__degreesignC as favorable helped significantly in disease development Incidence of powdery mildew was recoded low when the temperature decreased from 35__degreesignC (Fig.3). So 34-35__degreesignC was found to be the optimum Temperature for the development of powdery mildew that influenced the conidia__ampersandsign#39;s ability to germinate. Under these conditions their highest germination rate was 72% to exclude a change in the germination rate of conidia as a result of diurnal rhythm, inoculated plants were cultivated at a daily rhythm of 24__degreesignC/14__degreesignC. Conidia that development under these conditions already germinated at 15__degreesignC and reached their highest rate of germination (80%) at 20__degreesignC (Fig. 3). This germination rate was equivalent to that of conidia developing at a constant temperature of 25__degreesignC. This temperature range falls within the summer range in the AL. Gabel, AL. Akter region, when powdery mildew starts to appear on Cucumber crops. Conidia germinate best at a relative humidity of 97-100% [1], but not below, indicating that they require moist air to germinate [4] who demonstrated that germination decreased or was delayed in the presence of free water our the result is an agreed also with [18]. Who found that conidial germination of powdery mildew of cucumber was maximum at a temperature of 25__degreesignC, 100__degreesignC/R.H. These laboratory results may partially explain our field observations that powdery mildew symptoms appear first on leaves under dense canopies where the relative humidity is usually high. It has also been reported that cucurbit powdery mildew develops better in shade than in full sunlight [4, 11]. Development of mildew in relation to weather factors Weather conditions play an important role in the appearance and rapid build-up of especially the maximum temperature [28] found sunshine to be more important than R.H. for the development of powdery mildew on mung bean in Madhya Pradesh [28] while studying the role of weather factors for the development of powdery mildew on urd bean in Rabi season found maximum temperature between 21.0-26.1__degreesignC as favorable helped significantly in disease development [45] studied the development of powdery mildew and fund that early October sowing favors more disease development due to favorable weather conditions It has also been reported that cucurbit powdery mildew develops better in shade than in the full sunlight [4, 11]. Lack of information on the effect of humidity on the development of different stages of powdery mildew keeps us from knowing whether the phenomena described for E.cichrocerum.

Temperature and relative humidity appeared to influence the rate of growth of germ tubes after germination more markedly than percentage germination. The maximum length of germ tubes occurred between 15 to 30__degreesignC at temperatures 15, 30__degreesignC.with relative humidity 94 and 100% in the study. The shift in germination rate according to the temperature during spore development suggests that the fungus adjusts to the temperature, i.e., if the spores develop at low temperature, their germination rate at low temperature is higher than that of spores that develop at high temperatures. If, on the other hand, spores develop at high temperatures, their germination is reduced at low temperatures. At very high temperatures, germination occurs, but only at a rather low rate.

Our findings coincide largely with the results of other authors, so it can be concluded that natural conditions can be simulated in a climate chamber and reliable data can be obtained on the impact of various factors on the development of the fungus.

DECLARATIONS

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflicts of Interest

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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