MOLECULAR CHARACTERIZATION OF PENICILLIUM EXPANSUM ISOLATED FROM GRAPES AND ITS MANAGEMENT BY LEAF EXTRACT OF CHENOPODIUM MURALE

Article History Received: January 19, 2021 Revised: March 11, 2021 Accepted: March 16, 2021 Penicillium expansum Link causes an economically important postharvest blue mold disease in a number of fruits and vegetables. In the present study, this fungus was isolated from rotted grapes and identified on morphological basis. Identification of the pathogen was further confirmed on molecular basis by using four different primer pairs namely ITS, β-tubulin, CMD and CF under accession numbers MN752155, MN787831, MN787832 and MN787833, respectively. Leaf extract of Chenopodium murale was assessed for its potential to control in vitro growth of P. expansum. For this purpose, leaves were extracted in methanol and after evaporation of the solvent, the resulting extract was successively partitioned with n-hexane, chloroform, ethyl acetate and n-butanol followed by antifungal bioassays with different concentrations (1.562 to 200 mg mL-1) of each organic solvent fraction. Although all the fractions controlled the fungal pathogen to variable extents, however, n-butanol fraction showed the highest antifungal activity causing 45–86% reduction in the biomass of P. expansum. Ethyl acetate fraction was also highly antifungal and reduced fungal biomass by 44–81%. Chloroform and n-hexane fractions were comparatively less effective and reduced biomass of P. expansum by 30–72% and 11–44%, respectively. This study concludes that ethyl acetate and nbutanol fractions are highly antifungal in nature against P. expansum.


INTRODUCTION
Grape (Vitis vinifera L.) is the first fruit berry that has a long history of cultivation by humans (Morata et al., 2017). It is cultivated on commercial basis in more than 75 countries with an average production of more than 23 million metric tons (FAOSTAT, 2020). It is a domesticated deciduous woody vine having numerous uses for producing table fruit, jelly, juice, wine, jam, vinegar, raisins and grape seed oil (Venkitasamy et al., 2019). It is considered as a non-climacteric type of fruit having green, red or black colors that grows well in a cluster of 20 to 270 (Migicovsky et al., 2017). It is enriched with essential nutrients such as minerals, vitamins, edible fibers, carbohydrates and phytochemicals. In grapes, polyphenols are considered as the most important phytochemicals because they possess many health-promoting benefits and biological activities (Cheng et al., 2020). In addition, grapes are a rich source of antioxidants such as flavones, anthocyanins, linalool, geranial, tannins and nerol (González-Barreiro et al., 2014). Due to its highly perishable nature, grape production is largely affected by a wide range of fungal pathogens (Kassemeyer, 2017). Among them, Penicillium is one of the most common groups of fungi that attacks on fresh table grapes during packaging and causes significant postharvest decay losses (Wang et al., 2019). In recent years, Penicillium expansum has become the main pathogen responsible for blue mold disease of grape in Pakistan. It represents a serious economic concern to the grape industry causing up to 60% of decay in stored fruit (Ghuffar et al., 2018). It produces several toxic compounds such as chaetoglobosins, citrinin and patulin that affect the quality of processed products (Tragni et al., 2021). P. expansum conidia typically penetrate through wounds and propagate on fruit surface even at below 0°C (He et al., 2019).
Although synthetic fungicides such as fluxapyroxad, cyprodinil and thiophanate methyl are used commonly for the control of postharvest losses by P. expansum, however, their application on large scale has compromised their efficacy by developing resistance in pathogen populations (Samaras et al., 2020). Public health concerns have stimulated the search for alternative control strategies for the effective management of P. expansum. In nature, plants are considered as a rich source of volatile compounds that are biologically active against fungal pathogens (Akhtar et al., 2020;Javaid et al., 2020;da Silva et al., 2020). Many studies have focused on C. murale for the effective control of phytopathogens (Naqvi et al., 2019;Naqvi et al., 2020). It belongs to family Chenopodiaceae. It is one of the fast-growing erect annual herbaceous weed natives to Europe (Farhan et al., 2019). Now, it is widespread to various parts of the world including Pakistan where it grows on different types of habitats. It grows abundantly in winter and early summer along the roadsides, cross walks, damp and waste places (Al-Batsh and Qasem, 2020). It is enriched with diterpenoids, terpenoids, flavonoids, glycosides, sterols, sesquiterpenes, glucosinolates, alkaloids, coumarins and phenolic acids with potent antifungal activities (Belmaghraoui et al., 2018). Therefore, the aim of this study was to identify P. expansum on molecular basis and to utilize the leaf extracts of C. murale for effective management of this pathogen of grapes.

MATERIALS AND METHODS Isolation and characterization of P. expansum
Infected grapes with light brown spots were collected from Lahore, and surface sterilized in 3% sodium hypochlorite solution for 1 min followed by washing with autoclaved water. The diseased area was carried out by using a needle and placed on freshly prepared malt extract agar. The inoculated plates were kept at 30 °C for five days. The obtained colonies were re-isolated to get pure cultures and studied on the basis of macroscopic and microscopic features at different magnifications under light microscope (Vico et al., 2014).

Molecular identification using PCR
The genomic DNA of the isolated fungus P. expansum was isolated by CTAB method following the protocol described by Doyle and Doyle (1990). The conventional PCRs were performed by using ITS, β-tubulin, CMD and CF primer pairs (Table 1). The obtained PCR products were analyzed by MiSeq Illumina sequencing, USA for bidirectional sequencing and then submitted to NCBI database for BLAST alignment search.

Preparation of extracts
Fresh C. murale plant leaves were collected from Punjab University, Lahore. The collected leaves were washed under running tap water, shade dried and grinded into a fine powder. The powdered leaves (1 kg) were dipped in methanol (5 L) for two weeks at room temperature and then passed through a filtration process by using two layers of filter paper. The obtained material was run-on a rotary evaporator to gain a thick gummy leaf extract. The crude extract was mixed in autoclaved water (200 mL) and partitioned through a separating funnel with nhexane (4 × 500 mL), chloroform (200 mL), ethyl acetate (200 mL) and n-butanol (100 mL). The resultant solvents were run on rotary evaporator in order to get thick gummy material of each fraction (Banaras et al., 2020).

Antifungal bioassays
A mass of 1.2 g of each fraction of leaf extract was dissolved in DMSO (1 mL) followed by the addition of malt extract (ME) broth to raise the final volume up to 6 mL (stock solution) of 200 mg mL -1 concentration. The lower concentrations viz. 100, 50, 25, 12.5, 6.25 and 3.125 mg mL -1 were prepared by serial double dilution of the stock. A control set was also prepared without plant extract and each treatment was replicated thrice. Mature culture of P. expansum was used to prepare spore suspension and each glass tube was inoculated with 20 µL of it and left to stand at 30 °C for 7 days. After that, fungal mats were filtered on pre-weight filter papers, dried and weighed .

Statistical analysis
Data related to biomass of the P. expansum was analyzed by ANOVA and treatment means were separated by applying Tukey's HSD test at 5% level of probability using software Statistix 8.1.

Morphological identification of P. expansum
Morphological features showed that the mature colonies of the fungus were circular and rapidly growing attaining a diameter of 5 cm at 30 °C in 7 days. The young colonies were off white in color, which upon maturity turned into light to dark green color whereas the reverse side of the plate was pale yellow to greenish in color (Figure 1). The observed conidia were 3.25 to 3.71 × 2.56 to 3.28 µm in size, elliptical in shape, smooth surfaced having thin walls, attached with branched conidiophores. The germinated conidia form germ tubes. On the basis of morphological studies, the examined isolate was identified as P. expansum.

Molecular characterization of P. expansum
For more accuracy, molecular tools were used for the precise characterization of P. expansum as only microscopic studies are not sufficient due to huge similarities among the Penicillium species. Therefore, a total of four primer sets viz. ITS, β-tubulin, CMD and CF were used for the identification of P. expansum genomic DNA under MN752155, MN787831, MN787832 and MN787833 accession numbers, respectively with 99-100% similarities with already deposited specimens in the GenBank. As expected, the amplified PCR products generated specific bands of the appropriate sizes as shown in Figure 2. amplified PCR product, (5): CF1/CF4 amplified PCR product.

Screening bioassays
Four fractions of C. murale leaf extract were used against P. expansum that showed a significant suppressive effect on the growth of the targeted pathogen. Although all the fractions variably controlled the fungal pathogen, however, n-butanol fraction showed the highest antifungal activity causing 45-86% reduction in P. expansum biomass over control. Likewise, different concentrations of ethyl acetate fraction noticeably reduced the fungal biomass by 44-81%. The other two fractions viz. chloroform and n-hexane were comparatively less effective and reduced biomass of P. expansum just by 30-72% and 11-44%, respectively (Table 2).

DISCUSSION
In the present study, P. expansum was found as the causal agent of blue mold disease of grapes on both morphological and molecular bases. Blue mold decay is caused by many Penicillium species such as P. expansum, P. chrysogenum, P. rugulosum, P. polonicum, P. commune, P. italicum, P. digitatum, P. crustosum, P. solitum and P. verrucosum (Chen et al., 2017;Papoutsis et al., 2019;Duduk et al., 2021). Among these, P. expansum is the most virulent strain that produces toxins and causes blue mold decay of peach (Jiao et al., 2018), pear (Zhou et al., 2018), apple (Abdelhai et al., 2019), grapes (He et al., 2019) and quince (Luciano-Rosario et al., 2020). As the pathogen has a wide host range, therefore, its management is very critical. For this, different fractions of C. murale leaf extract were used and the findings revealed that all the fractions had antifungal activities. Earlier, Naqvi et al. (2019) reported that different fractions of C. murale leaf extracts significantly reduced the growth of Fusarium oxysporum f. sp. lycopersici. Abdel-Wahhab et al. (2020) reported the presence of antioxidants, flavonoids and phenolic compounds with strong antimicrobial activities that might be responsible in reducing the growth of targeted fungal pathogen. C. murale extract also suppressed growth of Alternaria solani and Penicillium digitatum (Qasem and Abu-Blan, 1995) as well as Macrophomina phaseolina (Amin and Arshad, 2007). Antifungal activity of C. murale leaf extract could be due to presence of hexadecanoic acid, methyl ester; palmitic acid; phytol; β-sitosterol and methyl linolenate (Naqvi et al., 2019).
The present study concludes that P. expansum is the causal agent of blue mold disease of grapes in Pakistan. Being a post-harvest pathogen, it produces toxins and contaminates the food products during packing and processing. For its management, leaf extract of C. murale was used that significantly reduced the growth of targeted pathogen. Further studies regarding methods of application and mechanism of actions of the C. murale leaf extract, and identification of effective antifungal compounds are necessary for effective control measures of this pathogen.