Isolation and Identification of Penicillium italicum from Iraqi Citrus Lemon Fruits and its Ability Manufacture of Silver Nanoparticles and their Antibacterial and Antifungal activity

 

Zainab K. Taha¹, Summaya N. Howar¹*, Ghassan M. Sulaiman²

¹Biology Department, College of Education for Pure Sciences/Ibn al-Haitham, University of Baghdad,

Baghdad, Iraq

²Biotechnology Division, Applied Science Department, University of Technology, Baghdad, Iraq

 

ABSTRACT:

Recently, attention to the manufacture of nanoparticles has been vital as a result to develop safe, low cost-effective and environmentally friendly methods for nanoparticle manufacturing techniques. In this study, Penicillium italicum was isolated and diagnosed from Iraqi citrus lemon fruits using various media including PDAs, MEA, CDA, Nitrite sucrose (no2) Agar, CYA and YES. Isolates were cultured at different temperatures of 5, 25 and 37°C to diagnose the morphological characteristics of P. italicum, and then using P. italicum in the production of silver nanoparticles using X-Ray diffraction and Atomic Force Microscope. The antifungal and antibacterial activity was determined against Cladosporium sp, Monilia sp., Fusarium sp., Candida glabrata and Klebsiella pneumonia. The results have been shown an inhibition effect of silver nanoparticles produced by P. italicumon the growth of studies bacteria and fungi in dose-dependent manner.

 

 

 

INTRODUCTION:

Nanotechnology technique has been used in several fields including medicine, Pharmaceutical industries, Agriculture, foods production, water technology, environmental protection, genetic engineering, genes and many applications in other fields. Nanoparticles are characterized with mechanical, thermal, chemical, biological and other properties which are different from the basic materials due to their large surface area to the volume ratio and quantum effect¹. To date, many physical and chemical strategies have been used to production nanoparticles^2,3. However, the focus was on the toxicity of chemical agents that used in the manufacture of AgNPs, so it was necessary to develop safe strategies to produce AgNPs without using hazardous substances to human health and the environment.

 

 

Compared to conventional production methods, biological systems provide innovative ideas for the production of nanomaterials⁴. Interestingly, fungi have been attracted in various research especially in the production of metallic nanoparticles due to their durability and bioaccumulation of minerals⁵ and also due to the dissociation of fungi from the medium and their effectiveness in the secretion of extracellular enzymes which can produce enormous of enzymes⁶. In this research, P. italicum was isolated from citrus lemon fruit and then used in the biosynthesis of silver nanoparticles. This method is an alternative to chemical and physical methods because it is very easy, non-toxic, safe to the environment and inexpensive, and also the availability of fungi is an important property to use fungi in the production of nanoparticles⁷. P. italicum or what is known as blue mold is one of the important causes of post-harvest citrus rot. The fungi of this species are live for long time and also are stable in adverse conditions; it is the most ferocious species on citrus fruits where it is can cause damage even if stored at low temperatures of about 5°C⁸. In this study, P. italicum was isolated and identification from Iraqi citrus fruits and its susceptibility to the production of AgNPs was determined and also the antifungal and antibacterial activity against K. peneumoniae was identified.

 

MATERIALS AND METHODS:

Materials:

Potato Dextrose Agar (PDA), Malt Extract Agar, and Czapek Dox Agar were purchased from Himedia (India). AgNO₃ which purchased from CDH (India), and Ethyl alcohol.

 

Living microorganisms used in the study:

Klebsiella pneumoniae and Candida glabrata were obtained from the service laboratory at the Faculty of Education for Pure Sciences (Ibn Al-Haitham), university of Baghdad, Iraq. Filamentous fungi including Cladosporum sp, Fuserium sp, Monilia sp was obtained from the advanced fungus laboratory located in the same faculty.

 

Isolation and diagnosis of fungi:

Penicilliumitalicum was isolated and identification from Iraqi citrus lemon fruits that collected from Iraqi markets; isolation and diagnosis were done in the postgraduate lab of fungi in Faculty of Education for pure sciences (Ibn Al-Haitham).

 

Collection of citrus lemon fruits and treatment:

Samples of mold citrus lemon fruits were collected from the markets in Baghdad. They were brought into the laboratory and washed with distilled water. They were sterilized by dipping for 2 minutes with sodium hypochloride solution (5%), then washed three times with sterile distilled water and dried using sterile filter papers and later used to isolate fungi.

 

Isolation and identification of causes of mold:

A 1-2 cm of each sample was taken from the edge of the infected spots of sterile and washed fruits and placed upside down on PDA medium that contains Chloramphenicol to prevent the growth of bacteria and kept at 25°C for 5-7 days⁹. The developing colonies were purified and diagnosed using the phenotypic and other morphological characteristics¹⁰. The growth of colonies was compared in different media including  Potato Dextrose Agar (PDA), Malt Extract Agar, Czapek Dox Agar Yeast, Nitrite Sucrose Agar which consists of 0.3gNaNO₂, 0.3g sucrose, 0.13g K₂HPO₄, 0.05 g MgSo₄, 0.05g KCl, 0.001 g FeSo₄, 0.0005g CuSo_4, 0.001g ZnSo₄, 2g agar, and 25 ml glycerol, Yeast Extract Sucrose Agar (YES) 2 g Yeast extract, 15 g Sucrose, 0.05 MgSO₄, 0.0005 g CuSO₄, 0.001 ZnSO₄, 2g Agar 2g¹¹ Czapek Yeast Auto lysate which consists of 0.3g NaOO3, 0.5g Yeast Extract, 3g Sucrose, 0.13g K₂HPO₄, 0.05g MgSo₄, 0.05gKCL, 0.001g FeSO₄, 0.005gCuSO₄, 0.001g ZnSO₄, 1.5 Agar¹² and the diameter of colonies was measured and microscopic properties were determined after 7 days.

Production of biomass of Penicillium italicum:

P. italicum was cultured in PDA medium for 7 days, and then a piece of 5-8 mm(one or two pieces) was taken by using platinum hole and placed in 200mLof liquid sterile medium which consists of 7g KH₂PO₄, 2g K₂HPO₄, 0.1g MgSO₄, 1g (NH₄)₂SO₄, 0.6 Yeast extract, 10g Glucose¹³ in the shaking incubator at 25°C with the speed of 130 rpm for 7 days to obtain biomass from the fungal Filamentous of P. italicum.

 

Preparation of Bioactive Nanomaterial solution:

The biomass of P. italicum was filtered using filter papers (Whatman No.1) and then washed at least three times to remove liquid residue with distilled water. AgNO3 solution (1.5 mM) was added with ratio of 100 ml to 10g of fungi biomass and placed in the darkness for 96 hours in the shaking incubator at 37°C with speed of 150 rpm.

 

Characterization of silver nanoparticles:

The silver nanoparticles were characterized by X-ray diffraction technique. Drops of silver particles solution were dropped onto a glass slide and dried at 50°C after being measured in XRD 6000 to detect the type of metal produced as well as its crystalline volume. For Atomic Force Microscope test, the sample prepared by dropping a few drops of the biosynthesized silver nanoparticles solution onto a glass slide and dried at 50°C. The Atomic Force Microscope is used to determine the topographic details of the crystalline surface for the granule, surface roughness and granular size. This examination was conducted at the Ministry of Science and Technology/Materials Research Department.

 

Biological effect on Filamentous fungi:

A 100μL of silver nanoparticle solution with a concentration of 25, 50, 100 and 200 μg mL^-1 was placed in Petri dishes of PDA medium and left for a period of time. Then, a piece with 5 mm in diameter was taken from the fungal growth of Cladosporum sp., Fusarium sp. and Monilia sp. at 7 days. Disc was placed in the middle of the fungi medium and incubated at 25°C for 7 days. Control dishes were placed at the same conditions without adding the silver nanoparticles solution and then the diameters of the growth colonies were measured.

 

Antibacterial and fungal efficacy assay:

The efficacy of silver nanoparticles was tested on Klebsiella pneumonia, (Gram negative bacteria) as well as Candida glabrata yeast by following Agar well diffusion method. Bacterial were cultured in the broth nutrient medium for 24 hours. The fungus was developed on the SD broth medium for 48 hours. A 100 μL of broth nutrient containing bacterial growth with (1 x 10⁵ cell mL^-1) was taken and cultured on Petri dishes containing nutrient agar medium, while for the fungi, 100 μL of the SD broth medium with (1 x 10⁵ cell mL^-1) was collected and spread on the surface of the petridishes containing SDA medium. The cultures were left for a short period and then holes with5 mm in diameter was drilled using sterile hole puncher. The holes for bacteria and fungi were filled with 60 μL of silver nanoparticle solution at different concentrations (400, 600, 800, 1000 μg mL^-1), while silver nitrate solution was used as a control agent, then all treated media were incubated at 37° C for 24 hours. After the growth period, the inhibition zone for the bacteria and fungi were measured¹⁴.

 

Figure 1:Pinecilliumitalicum growing on the iraqi lemon fruits

 

Figure 2: Identification the fungi Pinecillium italicum using different culture media at temperature 5, 25, 37^oC.

 

RESULTS AND DISCUSSION:

Identification of fungi that cause the rotting of citrus lemon fruits:

In this study, the fungal isolates that cause the mold for citrus lemon fruits are due to Penicillium which is the major causes of mold for this fruit. It is known that the identification of Penicillium at the level of the species is not easy due to the similarity between its multiple species in the phenotypic characteristics¹⁰. However, the identification has been determined under standard conditions including media, incubation period and temperature, morphological and microscopic characteristics, shape of conidia, as well as physiological properties including diameter of colonies and their characteristics on the various culture media, all of these properties helps in the identification of Penicillium^10,15. Penicillium isolates were obtained from citrus fruits in this study (Fig. 1). It was cultured and examined in different media including Czapek, Potato Dextros Agar (PDA), nitrate, Yeast, Malt and Czapek yeast Auto Lysate (CYA) (Fig. 2).

 

The colonies of Penicillium appeared on the nitrate medium in small colonies with diameters ranged from 2.9-3.8 cm at 25°C, while at 5°C and 37°C no growth was observed. Moreover, the growth of Penicillium on PDA medium was clear at 25°C and the colonies appeared in a bluish-brown color and were filled with white-colored velvet flakes, while the color of the reflection on the medium was brown, and the diameter of colonies was 4.8-5.9 cm. The growth of colonies on the same medium at 5°C was very weak with diameter of 1.5-2.0 mm. However, no growth shown at 37°C. While, the growth of colonies on Malt medium was appeared good and quick at a 25°C. The colonies appeared with blue color in the center while the rest of the colony was with white filaments without spores, and the color of the reflection on the medium was light brown color; the colony diameter rate was 5.4-6.2 cm, while it did not exceed 2-3 mm at 5° C and no growth was seen at 37°C.

 

On CYA medium, the growth of colonies of fungi was developed at temperature of 25°C; the form of colonies were tightly packed with white borders and a bluish green color and the diameters of colonies averaged 5.4-8.3 cm. The color of the reflection on the medium was dark brown, while the diameter at 5°C did not exceed 1-1.5 mm and showed no growth at 37°C. As well as for growth on the Yeast medium, the growth was also good and quick at 25°C, where the colonies appeared in a bluish-blue color and in white packed form with diameters of 5.1-6.6 cm, and the color of reflection on the medium was yellow. However, the growth on the same medium at 5°C was weak and the diameter was 2-3 mm and no growth was found at 37°C. Interestingly, the growth on Czapek medium was moderated at 25°C where the colonies appeared in the center in white color and the rest of the colonies in a green color with white borders, and the color of reflection on the medium was light brown. The diameter of colonies was 2.9-4.1 cm, while the diameter of at 5°C was 2-3 mm and there is no growth was found at 37°C.

 

The findings of this study revealed that the model of growth rate and colony diameters showed a variation between isolates as well as variation in the composition and the formation of spores between isolates that growth on the same medium. These observations are in agreement with study of¹⁶. The microscopic examination found that the heads of conidiophores (penicilli) were monoverticillat or terverticillat (Fig. 3-A). Conidia were clearly large and ellipsoidal to cylindrical in shape (Fig. 3-B) and these results are consistent with the results of [10]. The mycelium was hyaline and septate, while, the conidial conidiophores have smooth walls (Fig. 3-C) and these results are in agreement with observation of¹⁷ in terms of microscopic properties of studied fungus. P. italicum isolates that isolated from the mold citrus lemon fruits in this study is consistent with the evidence that observed P. italicum is a common contaminant fungus and one of the most important pathogens for citrus fruits in storage, and this result is consistent with the results of¹⁸ that revealed Penicillium is the most common fungus found on citrus.

 

The Biosynthesis of silver nanoparticles:

After mixing the biomass of P. italicum with the silver nitrate solution and after 96 hours of incubation in the shaking incubator in the dark, a change in the color of the mixture was observed from yellow to brown in comparison to the control solution that containing the fungal biomass without silver nitrate solution (Fig. 4). This chromatic change is an initial evidence of the formation of nanoparticle particles due to the presence of surface plasmids, which is in agreement with studies of [19] and [20]. Moreover, the change in the color is due to the difference in the electronic density of nanoparticles due to the difference in volumes of nanoparticles²¹.

 

 

Figure 3:Pinecilliumitalicum under the light microscope, 3-A: heads of conidiophores, 3-B: Conidia, 3-C: The mycelium.

 

Figure 4: Color change, A: AgNO₃ only, B: Fungi biomass extract only, C: Fungi biomass extract and AgNO₃.Characterization of silver nanoparticles

 

X-ray diffraction (XRD):

The importance of using x-ray diffraction is to clarify the crystalline structure of the materials as well as to give a visual appearance of the granulesize and crystalline defects. X-ray diffraction test was performed on nanoparticles that prepared in the biological way (using of fungi to produce nanoparticles); X-ray results have been showed that the prepared nanoparticles have a multi and cubic crystallization structure, and the crystalline levels formed are (111), (200), (220) and (311) which correspond to the diffraction angles (38.08°), (44.27°), (64.41°) and (77.36°), respectively through Joint Committee on Powered Diffraction Standard (JCPDS card no. 96-901-3046) and this is in agreement with previous researches of[22] [23]. The results of XRD analysis of(Fig. 5) revealed that the highest peak of the diffraction was shown at the crystalline level (111)with diffraction angle (38.08°=20),then the intensity of the peak begins to decrease at level 200 with the diffraction angle (44.27°=20).Moreover , the decreases in the intensity was increased at levels (202) and (311) and at angles (77.36°, 64.41°=20)with increase in peak width in middle of distance. The granular size was calculated by using Scherrer equation and the results were shown in Table (1).

The Surface topography structure:

The Atomic Force Microscope (AFM) is used to study the surface topography structure; thus through microscopic analysis in this study it can study the effect of parameters on the properties of sedimentary material because estudying the surfaces of the materials membranes is important in understanding their distribution and the arrangement of atoms on the surfaces, and also to identify the homogeneity of properties. Moreover, the roughness rate and granular size can also be calculated through a special program as this measurement is more accurate than X-ray analysis and gives an illustration of the distribution of granular size on the surfaces (Fig 6).

 

The surface roughness rate of the membrane was found to be average42.510 nm, and the mean square root value was 54.240nm and this value is defined as the total Surface elevations and decreases are divided by the sum of the total number under the square root. This value is a proof of the roughness of the surface, so when the mean square roots increases that give evidence to the higher roughness rate and vice versa. In figure (6), the shape of the granules is spherical and the membrane is homogeneous and uniformly crystalline. The granular size rate was about 81 nm.

 

The biological activity against filamentous fungi:

In this study, using of silver nanoparticles as antifungal agents observed that these nanoparticles caused morphological changes including defects in the growth of the colonies where their growth was irregular that represented by increasing in fungal filaments and decreasing in the reproductive structures.

 

 

 

 

Figure 5: XRD analysis for sample.

 

Table 1: Crystalline size of silver nanoparticles

 

Figure 6: Grain size of silver nanoparticles

 

These morphological defects were increased with increasing of concentrations while no inhibition of the colonies diameter were seen, and this may be due to the fact that these fungi produce secondary metabolites such as terpenoids, ribosomal peptides, nonribosomal peptides and polyketides, which may interact with silver ion and produce a kind of resistance, these observation are in agreement with [24] results which found that silver nanoparticles have an effect on the phenotypic structure of fungal colonies after treating the medium with nanoparticles, see fig 7.

 

Figure 7: Effect of silver nanoparticles against Filamentous fungi A-Fuserium B-Monilinia c- Cladosporum

 

Diffusion method to determine the activity of fungi and bacteria

The antibacterial and antifungal effect of manufactured silver nanoparticles against pathogens infected the human body is estimated by measuring the inhibition zone compared to control group which is the silver nitrate AgNO₃where no inhibitory effect was shown on bacteria and yeasts in comparison to silver nanoparticles which its inhibitory effects were found at different concentrations (400, 600, 800, 1000μgmL^-1). This is due to the size and surface area of silver nanoparticles.

 

The highest inhibition concentration of silver nanoparticles for C. glabrata was at 1000 μg mL^-1in comparison to control where the inhibition zones were20 and 36 mm, respectively. These findings give evidence that increasing the concentrations caused increases in the rate of inhibition as shown in Fig. 7 which is consistent with ^{25, 26, 27}.

 

The high activity of nanoparticles is due to the size and surface area of ​​these particles which enable them to reach and bind with microbial DNA. Furthermore, silver nanoparticles are believed to bind to the cell membrane and release silver ions which in turn disrupt cellular membrane permeability and inhibit DNA replication; these nanoparticles also have a high surface area that enable them to connect and interact with microbial cells with high efficiency. The antibacterial effect is due to the release of silver ions from silver nanoparticles that remain in the cell membrane result in changes in the structure and then increase in the membrane permeability which in turn cell death²⁸.

 

 

A                                                B

Figure 8. Antibacterial activity assay against: A- Klebsiella pneumonia  B- Candida glabrata

 

Figure 9: inhibition percentage of silver nano-particles against Klebsiella pneumonia and Candida glabrata

 

CONCLUSIONS:

The observations of this study concluded that P italicum isolated from Iraqi citrus lemon fruits has the ability to produce nanoparticles have antibacterial and antifungal (against filamentous fungi and yeast), and also these nanoparticles may interact with other useful applications.

 

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Received on 15.09.2018           Modified on 17.10.2018

Accepted on 19.11.2018         © RJPT All right reserved