First report of Penicillium glaucum Link causing Penicillium rot of pear fruits Pyrus communis L. in Jammu and Kashmir, India

Pears Pyrus communis L. collected from different sites of Kashmir Valley were found infected with Penicillium glaucum causing Penicillium rot. The diseased fruits appears light tan to dark brown. The decayed tissue becomes soft, watery and the lesion has a very sharp margin between diseased and healthy tissues. Decayed fruit has an earthy, musty odor. The pathogen was isolated and cultured on PDA medium for further fungal morphological observation and confirming its pathogenicity according to Koch’s postulates. Results of morphological data and pathogenicity test showed that the pears were infected by Penicillium glaucum Link resulting in Penicillium rot of pears. To our knowledge, it is the first report of pear fruit rot caused by P. glaucum in India. Study was also undertaken for the management of Penicillium rot of pear with some fungicides. It was revealed from the study that different concentration of fungicides brought about significant reduction in the mycelial growth and spore germination of Penicillium glaucum under in vitro conditions. Amongst the tested fungicides, carbendazim proved highly effective in inhibiting the mycelial growth and spore germination of Penicillium glaucum followed by hexaconozole, bitertanol and myclobutanil respectively. Higher concentration proved effective than lower concentrations.


Introduction
Fruits are important sources of essential nutrients, vitamins, minerals and are a major source of complex carbohydrates, antioxidants and anti-carcinogenic substances which are important for human health. Pear fruits are rich sources of vitamin C, dietary fiber, malic acid, citric acid, quinic acid, αketoglutaric acid, succinic acid, lactic acid, glycolic acid, shikimic acid, glyceric acid 266 Parveen et al. Braz. J. Biol. Sci., 2017, v. 4, No. 8, p. 265-272. and mucic acid (Blattny, 2003;Colaric et al., 2005). Pear fruits are also known to have pharmacological properties like antiinflammatory, anti-tumour and antiallergic, due to presence of little amount salicylates and benzoates present in the fruit (Macheix et al., 1990). The fruit production is facing threats by many post harvest diseases, the principal cause being the fungal diseases. The pear fruits are more vulnerable to post harvest diseases due to their perishable nature, little shelf life etc. The threat of these diseases is influenced by the way these horticultural crops are handled and stored. The fungal rot diseases caused heavy losses to the fruits in storage, transit as well as in fields. As per literature fungal rot of fruits cause huge losses upto 30% to the growers in terms of yield (Barkai-Golan, 2001;Bhale, 2011) and therefore, needs proper management in storage and under field conditions The aim of the present study was to identify the fungal pathogen associated with pears in Kashmir Valley and to study the effect of different fungicides on the causal pathogen.

Materials and methods
To investigate the fungi which cause the rotting of pear fruits in Kashmir Valley, diseased pear fruits were collected in separate polythene bags from different fields, markets, godowns and storage houses of Kashmir valley. These samples were either used immediately or stored at 10 °C in the laboratory for different pathological studies. Small portions of rotted tissues were isolated aseptically from the diseased pear fruits and transferred to Potato Dextrose Agar (PDA) medium. Pure colony cultures were obtained by subculturing the fungal growth in separate Petri plates containing the same medium. The pathogen was identified by their morphological, reproductive and cultural characteristics (Ellis, 1971;Barnett and Hunter, 1972;Watanabe, 2002;Gilman, 2008). For pathogenicity, pathogens were re-inoculated after isolation onto the healthy pear fruits (Tomkin and Trout, 1931). Then all the fruits were kept in clean polythene bags and incubated at 25 + 2 °C for ten days. These pathogenicity tests were used for the identification of plant pathogens and to confirm the detection of a particular disease. Identification of the disease and the pathogen was done following Koch's postulates. Different parameters such as symptoms caused by these fungi on the healthy pear fruits, cultural characteristics of the pathogens and microscopic studies of the pathogens were studied.
In the present study an attempt was made to study the effect of some selected fungicides under in vitro conditions for the control of Penicillium rot of pears caused by Penicillium glaucum.

Methodology
Different concentrations (1,000 ppm, 800 ppm, 600 ppm, 400 ppm and 200 ppm) of fungicides viz. carbendazim, hexaconozole, bitertanol and myclobutanil were prepared in sterilized distilled water. These different concentrations of fungicides were evaluated for their effect on mycelial growth of rot causing fungi, Penicillium glaucum by food poisoning technique (Adams and Wong, 1991). Appropriate concentration (1 mL) of fungicide solution was mixed with autoclaved and cooled PDA (9 mL) just before pouring into Petri plates. The medium was then dispensed uniformly into 90 mm diameter Petri plates and inoculated with 5mm mycelial disc of the pathogen from 10 day old fungal culture. Three replicates were maintained for each concentration including control without any treatment. The Petri plates were incubated at 25 + 2°C and observations of the mycelial growth of test fungus were recorded after seven days of incubation. The percent inhibition in mycelial growth due to various fungicidal treatments at different concentrations was computed as follows: Mycelial growth inhibition (%) = Where dc = average diameter of fungal colony in control, and dt = average diameter of fungal colony in treatment group.
For studying the effect of fungicides on spore germination, spore suspension was prepared in sterilized distilled water. Equal volume of spore suspension and the fungicide solutions were mixed in a test tube and then shaken. The mixture then contained the particular concentration of test fungicide. In case of control spore suspension was mixed with equal volume of distilled water. A drop of the mixture (about 0.1 mL) was then placed in the cavity slide and these were incubated for 25±2 o C in a moist chamber to maintain enough humidity. Three replicates were maintained for each treatment including the control. The slides were examined after 24hrs by hand tally counts at different microscopic fields. Percent spore germination of each treatment was calculated by the formula given by Kiraly et al. (1974).

Percent spore germination =
No.of spores germinated Total no.of spores examined × 100

Results and discussion
It was observed from the present study that pears in storage are infected by the fungus, Penicillium glaucum Link resulting in Penicillium mold rot of pears. The casual pathogen was identified on the basis of symptoms caused by the fungus on pear fruits, cultural and microscopic characteristics. On the diseased fruits the decayed area appears light tan to dark brown. The decayed tissue becomes soft, watery and the lesion has a very sharp margin between diseased and healthy tissues (Figure 1a). Spores may appear on the decayed area, starting at the infection site. Decayed fruit has an earthy, musty odor. The causal agent was isolated from the diseased fruits and cultured on PDA medium at 24 + 2 °C. After 2-3 days the fungus produced yellowish green colonies (Figure 1b). Microscopic observation revealed that mycelium is septate, with branched conidiophores that are 70 µm-110 µm long, biverticillate (one stage branched). Metulae is 7 µm-10 µm long. Metulae forms conidiogenous cells called sterigmata or phialides. Sterigmata is 8.50 µm-10.95 µm long, and gives rise to conidia that are oval or globose and 1.50 µm-2.20 µm in diameter (Figure 1c).

Control of Penicillium glaucum Link causing Penicillium rot of pear with fungicides
In the present study, the effect of some fungicides and plant extracts were evaluated on Penicillium glaucum causing Penicillium rot of pear. Different concentrations of fungicides and plant extracts were evaluated for their efficacy on the mycelial growth and spore germination of the test fungi.

Effect of different concentrations of fungicides on the mycelial growth of Penicillium glaucum
It was revealed from the results (Table 1, Figure 2) that all the fungicides such as carbendazim, hexaconozole, bitertanol and myclobutanil at different concentrations, viz. 1000 ppm, 800 ppm, 600 ppm, 400 ppm and 200 ppm brought about significant reduction in mycelial growth of Penicillium glaucum compared to control. However, carbendazim and hexaconozole was found more effective in inhibiting the mycelial growth followed by bitertanol and myclobutanil at the same concentration. Other concentrations also caused significant reduction in mycelial growth. In different concentrations of carbendazim the inhibition in mycelial growth varies from 100%-89.21% and in hexaconozole the mycelial growth inhibition varies from 100%-87.07%, respectively. Likewise, the inhibition in mycelial growth varies from 100%-80.07% in different concentrations of bitertanol and in different concentrations of myclobutanil the inhibition in mycelial growth varies from 93.37%-72.52%, respectively.  , 1985). The numbers followed by same alphabets are not statistically different according to LSD test.

Effect of different concentrations of fungicides on the spore germination of Penicillium glaucum
It was revealed from the results (Table 2, Figure 3) that all the fungicides, viz. carbendazim, hexaconozole, bitertanol and myclobutanil at different concentrations 1,000 ppm, 800 ppm, 600 ppm, 400 ppm and 200 ppm caused significant reduction in spore germination of Penicillium glaucum. Amongst the fungicides hexaconozole and carbendazim at highest concentration (1,000 ppm) was found most effective in reducing the germination of spores followed by bitertanol and myclobutanil at the same concentration. The other concentrations also brought about significant reduction in spore germination but to lesser extent. The percentage inhibition in spore germination in carbendazim varies from 25.87%-0.00% in different concentrations. In hexaconozole the inhibition in spore germination varies from 26.58%-0.00% in different concentrations where as in bitertanol the germination of spores varies from 46.26%-15.14% and in myclobutanil the reduction in spore germination varies from 49.19%-18.18% in different concentrations.  , 1985). The numbers followed by same alphabets are not statistically different according to LSD test.

Discussion
It was clear from the results that pear fruits are attacked by Penicillium glaucum Link which is responsible for Penicillium rot of pear fruits. The occurrence of fungal rot of pears and other fruits due to various other fungi have been reported all over the world by several workers (Sive and Resinizky, 1987;Adikaram, 1988;Singh and Prashar, 1989; Braz. J. Biol. Sci., 2017, v. 4, No. 8, p. 265-272. Snowdon, 1990Bottcher and Pohle, 1991;Niklis, 1994;Lennox et al., 2004;Xiao and Boal, 2004;Spotts and Castagnoli, 2010;Bashir et al., 2012;Parveen and Wani, 2015;Parveen et al., 2016). Penicillium glaucum have been reported to cause rot of apples and pears in Greece (Giapanoglou et al., 1999). Although many species of Penicillium have been documented to cause fungal rot pears in India, this is the first report of occurrence of Penicillium glaucum on pear fruits in India as per literature survey.
In the present study some fungicides were evaluated for their antifungal activity against the identified fungus, Penicillium glaucum Link. From the results it is clear that all the tested fungicides proved highly effective in reducing the mycelial growth and spore germination of Penicillium glaucum. Amongst the tested fungicides, carbendazim proved highly effective in inhibiting the mycelial growth and spore germination of Penicillium glaucum followed by hexaconozole, bitertanol and myclobutanil respectively. In the previous work, similar findings were reported by Cole et al. (2005) Mancozeb, Rovral, Luna Experience, Aliette, Ortiva, Fenomenal and Ortiva to study their ability to inhibit the growth of fungal pathogens like Penicillium nordicum, Penicillium verrucosum, Verticillium dahlia and Cladosporium sps. and reported that all the fungicides were able to inhibit the growth of analyzed fungi.