Effect of climatic factors on fruit rots ( Alternaria alternata and Aspergillus niger ) of ber ( Zizyphus mauritiana Lamk . ) and their management systems

The present study aimed to study influence of temperature and relative humidity on fruit rots (Alternaria alternata and Aspergillus niger) of ber (Zizyphus mauritiana Lamk.) and find appropriate management option during storage period. Maximum damage by A. alternata was observed at 25°C while A. niger showed maximum severity at 30°C. Positive relationship was observed between relative humidity (RH) and fruit rot by both pathogens which showed highest severity at 100% RH. The pre-and postinoculation treatment of ber fruits with four vegetable oils, four plant extracts and four fungicides showed significant differences in effectiveness in controlling both pathogens. Neem oil and its leaf extracts significantly (p<0.05) reduced severity of fruit rots by both pathogens. Among chemicals, the carbendazim at 1000 ppm proved effective against Alternaria and Aspergillus fruit rot. Chemical fungicides reduced fungus growth significantly (p<0.05) better than botanicals; however, botanical also showed better response than control. The effectiveness of botanicals in post-harvest storage of ber can be used at commercial scale.


INTRODUCTION
Ber is a tropical and subtropical fruit native to the northern hemisphere and belongs to the genus Ziziphus of the family Rhamnaceae and order Rhamnales.The family has 50 genera and more than 600 species (Pareek, 2001) of which the species Z. jujube Mill (Chinese jujube or Chinese date), Z. mauritiana Lamk (Indian jujube or ber) and Z. spina-christi (L.) wild (Christ's thorn) are the most important in terms of distribution and economic significance.Ber (Ziziphus mauritiana Lamk) is one of the most important fruit crops grown in arid and semi-arid regions of India (Pareek, 2001).Ber fruits are rich in dietary minerals and natural antioxidants for people in arid regions.Its fruit products are getting commercial angle in different countries, particularly for their health benefiting properties (Vithlani and Patel, 2010).Ber fruit rots have been identified as major problems next to powdery mildew in arid and semi-arid regions.Many pathogens were identified as causing agents of fruit rots of ber in storages but Alternaria alternata and Aspergillus niger were two major inciting pathogens (Sharma and Majumdar, 1993).Though, reported losses from fruit rots were 2 to 15% but this goes high during congenial climatic conditions.
Alternaria and Aspergillus fruit rot are the most commonly occurring fruit rots in ber.Temperature and relative humidity affect fruit rot development and establishment of pathogen infection.For this disease, the congenial temperature ranges from 20 to 30°C and high relative humidity (>90%).However, the temperature and relative humidity affect the severity and pathogenicity of fruit rots caused by several pathogens that is, A. alternata, A. niger, Colletotrichum capsici, Drechslera australiensis, Fusarium moniliforme and F. solani (Datar, 1995).
Application of fungicides is a common management strategy to control the post-harvest pathogens on harvested fruits.Several fungicides have been effective in checking the storage rots of fruits and vegetables caused by different fungal pathogens (Singh and Thakur, 2005;Khokhar and Tetarwal, 2012), but, use of plant derived substances is attracting consumers and researchers for health factors.Plants rich in defence factors like seconddary metabolites reported to have strong anti-fungal, antibacterial properties (Reichling, 2010), therefore, could be used in post-harvest management of fruits and vegetables (Sable and Gangawane, 2010), but, information on their use was very meager, particularly on minor fruits like ber.Therefore, the present study was designed to determine congenial climatic factors (temperature and relative humidity) for two major pathogens that is, A. alternata and A. niger and to identify suitable botanicals (vegetable oils, leaf extracts) for management of fruit rots of ber in storage conditions.

Inoculum preparation and inoculation
The pathogens were isolated from the infected ber fruits collected from various market and orchards of Bikaner district of Rajasthan.Pieces of transitional zone tissues of diseased fruits were scooped out and surface sterilized with 0.01% HgCl2 solution for 1 to 2 min, followed by three washing in sterilized distilled water and transferred in petri-dishes containing potato dextrose agar medium (PDA).The crude cultures were incubated for growth at 26 ± 1°C and 95% relative humidity.The sporulating cultures were identified by microscopic examination for morphological characters of somatic and reproductive structures using reference isolates of A. alternata (Fr.)Keissler and A. niger van Tieghem.The isolated fungi were purified using single spore technique and then kept in a refrigerator on potato dextrose agar (PDA) slants for sporulation and further use (Chai et al., 1999).Healthy ripe fruits of ber were collected from main market of Bikaner, Rajasthan and washed with sterilized water.Excess water was removed and fruits were prepared for inoculation.Cork-borer wounding method was used for inoculation of the ber fruits.In brief, a hole of 2 × 2 mm size was made with sterilized cork borer and 2 mm disc cut of inoculum matrix of respective pathogens from inoculums petri-dishes was inserted in the hole by replacing the host pulp.
A piece of sterilized wet absorbent cotton was placed inside the surface sterilized polythene bags to create a micro-humidity chamber and mouth of the bags was loosely tied and incubated at 26 ± 1°C.

Environmental factors
The inoculated fruits were kept in polythene bags at four treatment levels of temperature that is, 20, 25, 30 and 35°C along with 95% relative humidity in four replications.Sterilized cotton swab placed inside the polythene bags to create a micro-humidity chamber.In another set, inoculated fruits were kept at six levels of relative humidity namely, 50, 60, 70, 80, 90 and 100% at 26 ± 1°C.The inoculated fruits kept in a desiccator which contained H2SO4 solution to produce a particular level of relative humidity, as suggested by Buxton and Mellanby (1934).The desiccators were sealed with grease and kept at 26 ± 1°C.The severity was recorded on the basis of percent infected area of fruit surface on 4 th and 8 th day of inoculation as described in Table 1.

Vegetable oil
Four vegetable oils namely, mustard oil, groundnut oil, castor oil and neem oil along with one control were used in four replications.In pre-inoculation, the fruits were first smeared with test oil and then inoculate after 12 h and vice-versa in case of post-inoculation treatment.The inoculated fruits were kept in the surface sterilized polythene bags with a piece of sterilized wet absorbent cotton to create a micro-humidity chamber and mouth of the bags was loosely tied and incubated at 26 ± 1°C.Fruit rot severity was recorded on 4 and 8 days after inoculation (DAI) as described in Table 1.

Leaf extracts
Another set of treatments with leaf extracts of Azadirachta indica A. Juss.(Neem), Vinca rosea L. (Sadabahar), Withania somnifera Dunal.(Ashwagandha) and Ocimum sanctum L. (Tulasi) and a control were tested against fruit rots.Leaf extracts were prepared by washing, air drying and homogenized for 5 min in sterile water (1:1, w/v).The mixture was filtered through muslin cloth and Whatmen No. 1 filter paper.In pre-inoculation treatments, the fruits were dipped in test extracts for 10 min and then inoculated.While post-inoculation treatments were applied after inoculation with A. alternata and A. niger; the interval between inoculation and treat-ment with plant extracts or vice-versa was 12 h.Proper controls were maintained and inoculation and observations were performed as per standard procedure (Mayee and Datar, 1986).

Chemical fungicides
The effectiveness of chemical control of fruit rots was tested using four commercially used fungicides namely carbendazim (50 WP), captan (50 WP), chlorothalonil (75 WP) and penconazole (10 EC) at 1000 ppm a.i. as in pre-and post-inoculation treatments against both pathogens separately.In pre-inoculation treatment, fruits were first dipped in the test fungicide for 5 min, air-dried and inoculated 12 h later while, 12 h after inoculation the treatments were applied in post-inoculation study.The experiment was arranged in completely randomized design.The procedures for inoculation and incubation of fruits and method of observations for fruit severity were recorded as described earlier (Mayee and Datar, 1986).

Statistical analysis
Data on fruit rots severity were recorded on the basis of percent fruit area infected.This was assessed with the help of an assessment key suggested by Mayee and Datar (1986).The analysis of variance was analysed using OPSTAT software (http//hau.ernet.in).

Environmental factors
Normally, ber fruits are stored at room temperature (24°C) and 50 to 60% RH (Pareek, 2001) which found to be congenial for the fruit rots causing pathogens.The present study shows that the temperature significantly (p<0.05)affected growth of A. alternata and A. niger (Figure 1a Pathogen severity index.

Severity (%)
Character 0.0 No infection, ruptured fruits remain free from fungus after inoculation, fruit remain edible after removing inoculated area 0.1-5.0 Fungus growth remain only in inoculated area (<5%), fruit remain edible after removing inoculated area 5.1-10.0 Fungus showed slight growth to adjoining tissues (5 to 10%), fruits can be eaten after removing affected area 10.1-25.0Fungus showed rapid growth and covered significant area (10 to 25%) of the infected fruits from inoculated area, fruit produce bad smell and not edible >25.0Most of the fruits surface (>25%) get affected and produce bad smell and not edible and b).
Fruit rot severity of A. alternata was recorded to be highest at 25°C (16.0 and 27.5% at 4 th and 8 th DAI, respectively) while it was low at 20°C (9 and 16%) and 35°C (8.75 and 14.25%) (Figure 1a).In case of A. niger, the fruit rot severity was highest at 30°C (18.5% at 4 th DAI and 28.0% at 8 th DAI) followed by 25°C (13.25% at 4 th and 22.75% at 8 th DAI) and it was lowest at 20°C (9 at 4 th DAI and 15% at 8 th DAI) (Figure 1a and b).Similar observations were made in other crop species where 20 to 30°C reported to be optimum range for rapid development of fruit rot in chillies caused by A. alternata, A. niger, C. capsici, D. australiensis, F. moniliforme and Fusarium solani (Datar, 1995).The study suggests storing ber fruits at or below 20°C for longer time.
The severity of fruit rots by both fungi was increased with the increasing levels of relative humidity (Figure 2a and b).Maximum severity of Alternaria fruit rot was observed at 100% RH (16.25% at 4 th DAI and 24.0% at 8 th DAI) which was significantly higher than 3.75% at 4 th DAI and 7.5% at 8 th DAI at 50% level of relative humidity.Similar observations were also recorded in case of Aspergillus rot in ber where fruit rot severity was highest at 100% RH at both 4 th DAI (21.0%) and 8 th DAI (26.0%) while significantly (p<0.05)low at 50% RH (Figure 2a).Fruit rot severity caused by A. alternata and A. niger increased from 4 th to 8 th day of inoculation with increase in levels of humidity (Figure 2a) but it showed de-clining rate of increase (Figure 2b).It may be due to high level of initial severity by A. alternata and A. niger at higher levels of RH than 50% RH level.It might be due to enhanced conidial germination, infection and decreased host resistance at higher relative humidity (Oladiran and Iwa, 1993).However, significant reduction in relative humidity affects the quality of ber fruits in storage (Pareek, 2001); therefore, ber fruits should be stored at 50 to 60% for maintaining shelf life as well as reduce the chances of fruit rot development.

Vegetable oils
The present study showed that pre-and postinoculation coatings of vegetable oil on ber fruit significantly (p<0.05)reduced the severity of fruit rots caused by A. alternata and A. niger.Neem oil was most effective, but was at par with groundnut oil, and was significantly (p<0.05)better over castor and mustard oils in reducing the fruit rot severity by Alternaria fruit rot in pre-inoculation treatment (Table 2).The severity of Aspergillus fruit rot was also reduced by neem and castor oils but its efficiency was significantly (p<0.05)higher that groundnut and mustard oils.The post-inoculation treatments revealed that neem oil treatment was significantly better than mustard and groundnut oil in reducing the severity of fruit rot due to A. alternata (Table 2).Neem oil also provided effective control of Alternaria fruit rot as post-inocu lation agent compared to other tested oils.The oils have used commercially for storage of different fruits (Sonkar et al., 2009;Khokhar and Tetarwal, 2012) and present study indentified the potential vegetable oils for extending the storage life of pathogen free ber fruits.These oils can be used as pre-or post-inoculation treatments for effective protection against both Alternaria and Aspergillus fruit rot.

Effect of leaf extracts
The biodegradable and non-hazardous nature of botanicals increased their acceptance in postharvest management of fruit and vegetables (Srivastava and Lal, 1997;Dargan and Saxena, 2002).The results from four botanical extracts on fruit rots severity are presented in Table 3. Preand post-inoculation treatments of fruits with neem leaf extract was found most effective against both Alternaria and Aspergillus fruit rots.O. sanctum was also found to be effective treatment against these two post-harvest rots.While the performance of W. somnifera extract as pre-and post-inoculation treatment was relatively low against both rots.Similar reports were made by Meena et al. (2009) and Kumar et al. (2010).The variation in response of plant extracts may be due to differences in phytochemical profiles of tested plants and presence of anti-fungal compounds in leaf extracts (Cowan, 1999).

Effect of fungicides
Several kinds of synthetic fungicides had been successfully used to control the post-harvest decay of fruits and vegetables (Adaskaveg et al., 2004;Kanetis et al., 2007).But, chemical fungicides are acceptable in field level crop protection practices but their use at storage level in fruit and vegetables is discouraged for consumer and environmental benefits.However, present study tested four different fungicides against Alternaria and Aspergillus fruit rots in ber and found comparatively better than botanicals (Table 4 and Figure 3).Among organic protectants, vegetable oils found effective than plant extracts against A. alternata while A. niger but differences were non-significant.All fungicides were significantly (p<0.05)superior in reducing fruit rots severity as compared to control in both pre-and post-inoculation treat-Effect of relative humidity (RH; %) on fruit rots by Alternaria alternata and Aspergillus niger (a) and their progress in ber (b).ments at 4 th and 8 th days of inoculation.Carbendazim and chlorothalonil were found most effective against Alternaria fruit rot while carbendazim and penconazole for Aspergillus fruit rots.Similarly, different fungicides   namely; Bavistin, Benlate, Thiobendazole, Dithane M-45, Blitox and Aureofungin have been indentified against storage rots of apple and ber (Singh and Thakur, 2005;Khokhar and Tetarwal, 2012).A comparative analysis of three sets of anti-fungal agents indicates superiority of chemical fungicides, but their use is discouraged for consumer and environment reasons and also in organic fruits and vegetables.Therefore, the present study suggest use of vegetable oils of neem and ground nut and leaf extract of A. indica or O. sanctum as pre-infection treatment for keeping ber fruits for longer period.The same may be used for postinfection control of A. alternata and A. niger in ber and similar fruits.Bikaner market for sharing ber fruits for the study.

Figure 1 .
Figure 1.Effect of temperature on fruit rots by Alternaria alternata and Aspergillus niger (a) and their progress in ber (b).

Figure 3 .
Figure 3. of different treatments on fruit rot severity (mean values) of ber.

Table 2 .
Effect vegetable oils on fruit rot severity by Alternaria alternata and Aspergillus niger in ber.

Table 3 .
Effect of plant extracts on fruit rot severity (%) by Alternaria alternata and Aspergillus niger in ber.

Table 4 .
Effect of chemical fungicides on fruit rot severity (%) by Alternaria alternata and Aspergillus niger in ber.