INTERACTION OF PENICILLIUM WITH PLANTS

Penicillium belongs to the kingdom ‘Fungi,’ phylum ‘Ascomycota,’ class ‘Eurotiomycetes’ order ‘Eurotiales,’ family ‘Trichocomaceae,’ genus ‘Penicillium.' Penicillium is repeatedly referred to as Deuteromycetes, or Fungi imperfecti. The name Penicillium derives from the word “brush,” which denotes to the appearance of spores in Penicillium. There are over 300 species of Penicillium, and Penicillium chrysogenum (one of the species) is classified as a psychrotrophic microorganism, which has the best lipase enzyme activity. Moreover, it was also found that among all the other fungi studied in the artic tundra, Penicillium chrysogenum showed maximum production of lipase. Penicillium chrysogenum has the capability to produce alpha-amylase as it has high enzymatic activity. Secondary metabolites are also produced due to some component that is present in the genetical structure of the fungus. Species of Penicillium are omnipresent soil fungi favoring over cool and moderate climates, generally present wherever organic material is accessible. Saprophytic species of Penicillium and Aspergillus are among the best-recognized representatives of the Eurotiales; besides, they mainly feed on organic decomposable substances. Penicillium is filamentous fungi and has split conidiospores. Round conidia are present and are unicellular. Cell walls of Penicillium species are mainly composed of Glucans. Penicillium species tend to have minor liyphae due to which the protoplasmic movement challenging to perceive. The small liyphae also lead to reduced peripheral growth zones. Penicillium spores are capable of getting wet though they have a hydrophobic surface, and this is necessary for germination to occur. Penicillium is osmotolerant, making sense that although they nurture better with high water levels, and they are able to bear low water potential (Sharma et al., 2017).

Penicillium species are heterotrophic. The pathogenic species feed off of the natural product they wreck. Penicillium produces asexually and is incapable to have spomlation when submerged. However, they begin their reproduction straightforwardly when the hyphae arise into a gas phase. No species exhibit this exact mode of reproduction, and each fungus is classified on the bases of its reproduction. For example, in some species, conidia are borne on phialidies, which assembly out of the conidiophore. In others, the conidiophore bears metulae, where phialides are borne. In other fungi, the conidiophore may branch out before bearing metulae. Branching may or may not be symmetrical, liable on species. Spomlation is not encouraged by changes in oxygen, carbon dioxide, or water loss. Instead, it is associated with the modification in the physical environment at the hyphal surface (Jeevitha et al., 2012).

Penicillium fungi are versatile and opportunistic. They are post-harvest pathogens. Penicillium species are one of the well-known causes of fungal decomposition in fruits and vegetables. P italicum and P digitatum are the most communal invaders of citrus fruits, while P expansum is identified to dose apples. P digitatum works by emancipating ethylene to accelerate ripening. It then asylums fhiit with green conidia initiating the fruit to shrink and diy out. P italicum causes slimy rot and harvests blue- green conidia. These species prefer cooler temperatures, which clarifies why they are frequently found on foods left too long in the refrigerator. Many species yield mycotoxins; for example, P expansum produces one termed patulin. Most of these species bear a resemblance to each other in color characteristics, decay style along with infection symptoms; they fall under a general group called blue mold. P expansum is one of the most destructive species. These fungi live a long time and are quite hard- wearing, even under contrary conditions. Occasionally, P italicum and P expansum will adhere to each other to create synnemata. Synnemata also occurs in Penicillum claviforme. Penicillium growth typically occurs as a result of wound infections in the crop. The most common treatment is to use fungicide on the harvested crop. Penicillium species attack more than just fruit. For example, Penicillium verrucosum breeds on cereal products (Lund et al., 2003).

IDENTIFICATION OF PENICILLIUM STRAIN

A common classification system is used for living organisms to identify species and genus, and in this system, species is the core unit. Taxonomic rank is classified as family, order, class, phylum, and kingdom. So basically, the fungi are classified over 300 species and can be identified on the bases of their morphological characters (visualizing colony) and microscopic characters (under a microscope). The macroscopic observation includes colony diameter, conidium color and reverses mycelium color, degree of spomlation, degree of sulcation, degree of wooliness, and the appearances or nonappearance of exudates. Surface characters include texture velvety to powdeiy; green, blue-green, gray-green, white, yellow, or pinkish on the surface, and the reverse characters are usually white to yellowish, sometimes red or brown (El-Fadaly et al., 2015). Hyphae septate, hyaline, conidiophores simple or branched. Phialidies grouped in brush-like clusters (penicillin) at the ends of the conidiophores; conidia unicellular, round to ovoid, hyaline or pigmented, rough walled or smooth and in chains, are some of the typical microscopic characters of PeniciUium spp. and molecular identification is done by isolating DNA of the fungi, purifying it, and going for 18S rRNA gene identification of them.

Mycologists used tools that were available to them at their time for identification, so it’s quite obvious that the features used for classifying the fungi have altered over time. Hence, the features that were used once, which were significant at that time seem to be unreliable with the time. The primary classification of fungi was done on macroscopic structures of the growth of fungi. However, certainly by the late 1800s, it had turned out to be clearer that fungi could occasionally show dissimilarity in form, depending on peripheral conditions, and clearly, it is futile to base a classification on any features subject to alteration by peripheral conditions. Hence, to overcome such superior dilemma confidence was shown on microscopic features and was considered for identification, since these were not modified by peripheral factors. Of course, this feature added a little value in identification. Figure 4.1 shows a few morphological characters of PeniciUium spp. and Figure 4.2 shows the microscopic structures of PeniciUium spp.

MOLECULAR IDENTIFICATION OF PENICIUIUM SPP. USING 18S rRNA GENE SEQUENCE ANALYSIS

18S rRNA gene identification is generally carried out preliminary for strain identification. Precise primers are designed for diverse fungal groups. However, owing to the massive diversity in fungi, true group- and species-specific recognition can be problematic to achieve by using selective primer-based PCR amplification unaided. A two-step recognition approach, by which a cluster of target DNAs from the amalgamated samples is selectively amplified which is latter followed by additional precise examination through probe hybridization that to a specific target in the PCR amplicon, can provide improved specificity besides sensitivity for environmental sample screening (Fujita et al.,2016). Currently, the product obtains after PCR are blotted on nylon membranes and then are further screened with oligonucleotide probes, each intended to be specific to a genus or a species. This level of detection of compassion is accurate in the rapports of environmental samples. The sequences of the oligonucleotide probe are explicit for quite a few groups of fungi with diverse detection possibilities that have been well-known (Visagie et al., 2014).

Morphological features of some (a-g) Penicillium spp. and(h) Trichoderma viride

FIGURE 4.1 Morphological features of some (a-g) Penicillium spp. and(h) Trichoderma viride.

Microscopic features of some (a-c) Penicillium spp. and (d) Aspergillus flavus

FIGURE 4.2 Microscopic features of some (a-c) Penicillium spp. and (d) Aspergillus flavus.

PCR amplification of 18S rRNA gene from the purified genomic DNA of any fungal isolate can be carried out by means of the following universal primers (forward primer 5’-GGAAGTAAAAGTCGTAACAAGG-3’ and reverse primer 5’-TCCTCCGCTTATTGATATGC-3’). Thermal cycler (PCR machine) conditions involved an initial denaturation step at 95°C for 5 min, trailed by 30 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min, and a final extension at 72°C for 1 min, followed by holding at 4°C. Amplified gene products are known as an amplicon, and they can be sequenced. Using the sequence data, the BLASTn search program (http:// wvw.ncbi.nlm.nih.gov) can be used to look for nucleotide sequence homology for strain identification (Thakor et al., 2016).

DNA barcoding is an efficient way to identify the eukaryotic organism, by employing a standardized short DNA sequence and a curated locus database connected to convincingly recognized vouchers (Hebert et al., 2003; Blaxter et al., 2005; De Salle et al., 2005; Schoch et al., 2012). After 18S rRNA gene sequencing, this remains the secondary method to identify. For fungi, the internal transcribed spacer (ITS) region of rDNA are recognized as the authorized barcode. ITS is the furthermost extensively sequenced marker for fungi, in addition, the universal primers are also available (Schoch et al., 2012). In Penicillium, it works glowingly for categorizing the strains into a species complex or else in one of the 25 sections, besides it every so often delivers a species identification. The International Commission of Penicillium and Aspergillus (ICPA), in combination with the publication of an updated recognized species list presented below, categorical to include GenBank accession numbers to Penicillium as reference barcode sequences for individually species when accessible.

p-tubulm (BenA) as the best possibility for a tributary identification marker for Penicillium but has a constraint that BenA like paralogous genes are also located in Aspergillus and Talaromyces genes that can be misleadingly amplified (Peterson, 2008; Hubka and Kolarik, 2012; Peterson and Jurjevic, 2013). Other than these. Calmodulin (CaM) or the second largest subunit of RNA polymerase II (RPB2) genes are also used as a tributary identification marker for Penicillium spp. Generally, a standard thermal cycle with an annealing temperature of 55°C is rummage-sale. For PCR amplification, amplification accomplishment is low for CaM, exclusively in sections Canescentia and Ramosa. In this circumstance, lowering the annealing temperature to 52°C gives good outcomes. The RPB2 amplification is more intricate. A touch-up PCR (50-52-55°C) with primer pair

5Feur and 7CReur are recommended for the best amplification. Once amplification is challenging, the substitute touch-up PGR (48-50-52°C) outline can be used and/or another primer pair 5F and 7CR. After amplification, these amplicons are sequenced. The sequences so obtained are compared with the other sequence of the same genes in the databank repository using the BLAST tool on NCBI. Using this technique, one is able to pursuit all sequences in the database, but so far, it has been noted that there are several unidentified and misidentified sequences in the database. From the NCBI homepage (http://www.ncbi.nhn.nih.gov/refseq/), the ITS, the RefSeq data set, is handy, and it is now available to use for the query ITS sequences in contradiction of a verified ITS database (Schoch et al., 2012).

 
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