Loading [MathJax]/jax/output/SVG/jax.js
ARTICLE   Open Access    

Endophytic mycobiota of wild medicinal plants from New Valley Governorate, Egypt and quantitative assessment of their cell wall degrading enzymes

More Information
  • The present study isolated and identified 32 species of endophytic mycobiota belonging to 18 genera associated with 8 wild medicinal plants collected from El-Kharga Oasis, New Valley Governorate, Egypt. Fusarium was the most common genus followed by Alternaria and Aspergillus. Convolvulus arvensis was the plant with the highest number of endophytes over the other plant species, while Moringa oleifera reported the lowest number of endophytes. In addition, the entomopathogenic fungus Beauveria bassiana; was recorded for the first time from leaves of Portulaca oleracea. One hundred and twenty-three isolates representing 32 species were screened for their abilities to produce pectinase, carboxy methyl cellulase (CMCase) and avicellase enzymes on sucrose free-Cz supplemented, individually with 1% pectin or 1% CMC or 1% avicel as a sole carbon source, respectively. Ninety-four isolates produced pectinase while 66 isolates produced cellulases. The quantitative assays of the three enzymes for high-producers were performed in submerged fermentation using sucrose-free Cz broth. Aspergillus was the superior in the production of the three enzymes with the potent strains were A. terreus AUMC 14287 for CMCase (22.0 IU/ml/min) and avicellase (47.868 IU/ml/min) and A. terreus AUMC 14278 for pectinase (225.43 IU/ml/min).
  • Grapevines are among the most widely grown and economically important fruit crops globally. Grapes are used not only for wine making and juice, but also are consumed fresh and as dried fruit[1]. Additionally, grapes have been increasingly recognized as an important source of resveratrol (trans-3, 5,4'-trihydroxystilbene), a non-flavonoid stilbenoid polyphenol that in grapevine may act as a phytoalexin. In humans, it has been widely reported that dietary resveratrol has beneficial impacts on various aspects of health[2, 3]. Because of the potential value of resveratrol both to grapevine physiology and human medicine, resveratrol biosynthesis and its regulation has become an important avenue of research. Similar to other stilbenoids, resveratrol synthesis utilizes key enzymes of the phenylpropanoid pathway including phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL). In the final steps, stilbene synthase (STS), a type II polyketide synthase, produces trans-resveratrol from p-coumaroyl-CoA and malonyl-CoA, while chalcone synthase (CHS) synthesizes flavonoids from the same substrates[4, 5]. Moreover, trans-resveratrol is a precursor for other stilbenoids such as cis-resveratrol, trans-piceid, cis-piceid, ε-viniferin and δ-viniferin[6]. It has been reported that stilbenoid biosynthesis pathways are targets of artificial selection during grapevine domestication[7] and resveratrol accumulates in various structures in response to both biotic and abiotic stresses[812]. This stress-related resveratrol synthesis is mediated, at least partialy, through the regulation of members of the STS gene family. Various transcription factors (TFs) participating in regulating STS genes in grapevine have been reported. For instance, MYB14 and MYB15[13, 14] and WRKY24[15] directly bind to the promoters of specific STS genes to activate transcription. VvWRKY8 physically interacts with VvMYB14 to repress VvSTS15/21 expression[16], whereas VqERF114 from Vitis quinquangularis accession 'Danfeng-2' promotes expression of four STS genes by interacting with VqMYB35 and binding directly to cis-elements in their promoters[17]. Aided by the release of the first V. vinifera reference genome assembly[18], genomic and transcriptional studies have revealed some of the main molecular mechanisms involved in fruit ripening[1924] and stilbenoid accumulation[8, 25] in various grapevine cultivars. Recently, it has been reported that a root restriction treatment greatly promoted the accumulation of trans-resveratrol, phenolic acid, flavonol and anthocyanin in 'Summer Black' (Vitis vinifera × Vitis labrusca) berry development during ripening[12]. However, most of studies mainly focus on a certain grape variety, not to investigate potential distinctions in resveratrol biosynthesis among different Vitis genotypes. In this study, we analyzed the resveratrol content in seven grapevine accessions and three berry structures, at three stages of fruit development. We found that the fruits of two wild, Chinese grapevines, Vitis amurensis 'Tonghua-3' and Vitis davidii 'Tangwei' showed significant difference in resveratrol content during development. These were targeted for transcriptional profiling to gain insight into the molecular aspects underlying this difference. This work provides a theoretical basis for subsequent systematic studies of genes participating in resveratrol biosynthesis and their regulation. Further, the results should be useful in the development of grapevine cultivars exploiting the genetic resources of wild grapevines. For each of the seven cultivars, we analyzed resveratrol content in the skin, pulp, and seed at three stages of development: Green hard (G), véraison (V), and ripe (R) (Table 1). In general, we observed the highest accumulation in skins at the R stage (0.43−2.99 µg g−1 FW). Lesser amounts were found in the pulp (0.03−0.36 µg g−1 FW) and seed (0.05−0.40 µg g−1 FW) at R, and in the skin at the G (0.12−0.34 µg g−1 FW) or V stages (0.17−1.49 µg g−1 FW). In all three fruit structures, trans-resveratrol showed an increasing trend with development, and this was most obvious in the skin. It is worth noting that trans-resveratrol was not detectable in the skin of 'Tangwei' at the G or V stage, but had accumulated to 2.42 µg g−1 FW by the R stage. The highest amount of extractable trans-resveratrol (2.99 µg g−1 FW) was found in 'Tonghua-3' skin at the R stage.
    Table 1.  Resveratrol concentrations in the skin, pulp and seed of berries from different grapevine genotypes at green hard, véraison and ripe stages.
    StructuresSpeciesAccessions or cultivarsContent of trans-resveratrol (μg g−1 FW)
    Green hardVéraisonRipe
    SkinV. davidiiTangweindnd2.415 ± 0.220
    V. amurensisTonghua-30.216 ± 0.0410.656 ± 0.0432.988 ± 0.221
    Shuangyou0.233 ± 0.0620.313 ± 0.0172.882 ± 0.052
    V. amurensis × V. ViniferaBeibinghong0.336 ± 0.0761.486 ± 0.1771.665 ± 0.100
    V. viniferaRed Global0.252 ± 0.0510.458 ± 0.0571.050 ± 0.129
    Thompson seedless0.120 ± 0.0251.770 ± 0.0320.431 ± 0.006
    V. vinifera × V. labruscaJumeigui0.122 ± 0.0160.170 ± 0.0210.708 ± 0.135
    PulpV. davidiiTangwei0.062 ± 0.0060.088 ± 0.009nd
    V. amurensisTonghua-30.151 ± 0.0660.324 ±0.1040.032 ± 0.004
    Shuangyou0.053 ± 0.0080.126 ± 0.0440.041 ± 0.017
    V. amurensis × V. ViniferaBeibinghong0.057 ± 0.0140.495 ± 0.0680.087 ± 0.021
    V. viniferaRed Global0.059 ± 0.0180.159 ± 0.0130.027 ± 0.004
    Thompson seedless0.112 ± 0.0160.059 ± 0.020nd
    V. vinifera × V. labruscaJumeigui0.072 ± 0.0100.063 ± 0.0170.359 ± 0.023
    SeedV. davidiiTangwei0.096 ± 0.0140.169 ± 0.0280.049 ± 0.006
    V. amurensisTonghua-30.044 ± 0.0040.221 ± 0.0240.113 ± 0.027
    Shuangyound0.063 ± 0.0210.116 ± 0.017
    V. amurensis × V. ViniferaBeibinghongnd0.077 ± 0.0030.400 ± 0.098
    V. viniferaRed Global0.035 ± 0.0230.142 ± 0.0360.199 ± 0.009
    Thompson seedless
    V. vinifera × V. labruscaJumeigui0.077 ± 0.0250.017 ± 0.0040.284 ± 0.021
    'nd' indicates not detected in samples, and '−' shows no samples are collected due to abortion.
     | Show Table
    DownLoad: CSV
    To gain insight into gene expression patterns influencing resveratrol biosynthesis in 'Tangwei' and 'Tonghua-3', we profiled the transcriptomes of developing berries at G, V, and R stages, using sequencing libraries representing three biological replicates from each cultivar and stage. A total of 142.49 Gb clean data were obtained with an average of 7.92 Gb per replicate, with average base Q30 > 92.5%. Depending on the sample, between 80.47%−88.86% of reads aligned to the V. vinifera reference genome (Supplemental Table S1), and of these, 78.18%−86.66% mapped to unique positions. After transcript assembly, a total of 23,649 and 23,557 unigenes were identified as expressed in 'Tangwei' and 'Tonghua-3', respectively. Additionally, 1,751 novel transcripts were identified (Supplemental Table S2), and among these, 1,443 could be assigned a potential function by homology. Interestingly, the total number of expressed genes gradually decreased from the G to R stage in 'Tangwei', but increased in 'Tonghua-3'. About 80% of the annotated genes showed fragments per kilobase of transcript per million fragments mapped (FPKM) values > 0.5 in all samples, and of these genes, about 40% showed FPKM values between 10 and 100 (Fig. 1a). Correlation coefficients and principal component analysis of the samples based on FPKM indicated that the biological replicates for each cultivar and stage showed similar properties, indicating that the transcriptome data was reliable for further analyses (Fig. 1b & c).
    Figure 1.  Properties of transcriptome data of 'Tangwei' (TW) and 'Tonghua-3' (TH) berry at green hard (G), véraison (V), and ripe (R) stages. (a) Total numbers of expressed genes with fragments per kilobase of transcript per million fragments mapped (FPKM) values; (b) Heatmap of the sample correlation analysis; (c) Principal component analysis (PCA) showing clustering pattern among TW and TH at G, V and R samples based on global gene expression profiles.
    By comparing the transcriptomes of 'Tangwei' and 'Tonghua-3' at the G, V and R stages, we identified 6,770, 3,353 and 6,699 differentially expressed genes (DEGs), respectively (Fig. 2a). Of these genes, 1,134 were differentially expressed between the two cultivars at all three stages (Fig. 2b). We also compared transcriptional profiles between two adjacent developmental stages (G vs V; V vs R) for each cultivar. Between G and V, we identified 1,761 DEGs that were up-regulated and 2,691 DEGs that were down-regulated in 'Tangwei', and 1,836 and 1,154 DEGs that were up-regulated or down-regulated, respectively, in 'Tonghua-3'. Between V and R, a total of 1,761 DEGs were up-regulated and 1,122 DEGs were down-regulated in 'Tangwei', whereas 2,774 DEGs and 1,287 were up-regulated or down-regulated, respectively, in 'Tonghua-3' (Fig. 2c). Among the 16,822 DEGs between the two cultivars at G, V, and R (Fig. 2a), a total of 4,570, 2,284 and 4,597 had gene ontology (GO) annotations and could be further classified to over 60 functional subcategories. The most significantly represented GO terms between the two cultivars at all three stages were response to metabolic process, catalytic activity, binding, cellular process, single-organism process, cell, cell part and biological regulation (Fig. 2d).
    Figure 2.  Analysis of differentially expressed genes (DEGs) at the green hard (G), véraison (V), and ripe (R) stages in 'Tangwei' (TW) and 'Tonghua-3' (TH). (a) Number of DEGs and (b) numbers of overlapping DEGs between 'Tangwei' and 'Tonghua-3' at G, V and R; (c) Overlap among DEGs between G and V, and V and R, for 'Tangwei' and 'Tonghua-3'; (d) Gene ontology (GO) functional categorization of DEGs.
    We also identified 57 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that were enriched, of which 32, 28, and 31 were enriched at the G, V, and R stages, respectively. Seven of the KEGG pathways were enriched at all three developmental stages: photosynthesis-antenna proteins (ko00196); glycine, serine and threonine metabolism (ko00260); glycolysis/gluconeogenesis (ko00010); carbon metabolism (ko01200); fatty acid degradation (ko00071); cysteine and methionine metabolism (ko00270); and valine, leucine and isoleucine degradation (ko00280) (Supplemental Tables S3S5). Furthermore, we found that the predominant KEGG pathways were distinct for each developmental stage. For example, phenylpropanoid biosynthesis (ko00940) was enriched only at the R stage. Overall, the GO and KEGG pathway enrichment analysis showed that the DEGs in 'Tangwei' and 'Tonghua-3' were enriched for multiple biological processes during the three stages of fruit development. We then analyzed the expression of genes with potential functions in resveratrol and flavonoid biosynthesis between the two cultivars and three developmental stages (Fig. 3 and Supplemental Table S6). We identified 30 STSs, 13 PALs, two C4Hs and nine 4CLs that were differentially expressed during at least one of the stages of fruit development between 'Tangwei' and 'Tonghua-3'. Interestingly, all of the STS genes showed increasing expression with development in both 'Tangwei' and 'Tonghua-3'. In addition, the expression levels of STS, C4H and 4CL genes at V and R were significantly higher in 'Tonghua-3' than in 'Tangwei'. Moreover, 25 RESVERATROL GLUCOSYLTRANSFERASE (RSGT), 27 LACCASE (LAC) and 21 O-METHYLTRANSFERASE (OMT) DEGs were identified, and most of these showed relatively high expression at the G and V stages in 'Tangwei' or R in 'Tonghua-3'. It is worth noting that the expression of the DEGs related to flavonoid biosynthesis, including CHS, FLAVONOL SYNTHASE (FLS), FLAVONOID 3′-HYDROXYLASE (F3'H), DIHYDROFLAVONOL 4-REDUCTASE (DFR), ANTHOCYANIDIN REDUCTASE (ANR) and LEUCOANTHOCYANIDIN REDUCTASE (LAR) were generally higher in 'Tangwei' than in 'Tonghua-3'at G stage.
    Figure 3.  Expression of differentially expressed genes (DEGs) associated with phenylalanine metabolism. TW, 'Tangwei'; TH, 'Tonghua-3'. PAL, PHENYLALANINE AMMONIA LYASE; C4H, CINNAMATE 4-HYDROXYLASE; 4CL, 4-COUMARATE-COA LIGASE; STS, STILBENE SYNTHASE; RSGT, RESVERATROL GLUCOSYLTRANSFERASE; OMT, O-METHYLTRANSFERASE; LAC, LACCASE; CHS, CHALCONE SYNTHASE; CHI, CHALCONE ISOMERASE; F3H, FLAVANONE 3-HYDROXYLASE; FLS, FLAVONOL SYNTHASE; F3'H, FLAVONOID 3′-HYDROXYLASE; DFR, DIHYDROFLAVONOL 4-REDUCTASE; LAR, LEUCOANTHOCYANIDIN REDUCTASE; ANR, ANTHOCYANIDIN REDUCTASE; LDOX, LEUCOANTHOCYANIDIN DIOXYGENASE; UFGT, UDP-GLUCOSE: FLAVONOID 3-O-GLUCOSYLTRANSFERASE.
    Among all DEGs identified in this study, 757 encoded potential TFs, and these represented 57 TF families. The most highly represented of these were the AP2/ERF, bHLH, NAC, WRKY, bZIP, HB-HD-ZIP and MYB families with a total of 76 DEGs (Fig. 4a). We found that the number of downregulated TF genes was greater than upregulated TF genes at G and V, and 48 were differentially expressed between the two cultivars at all three stages (Fig. 4b). Several members of the ERF, MYB, WRKY and bHLH families showed a strong increase in expression at the R stage (Fig. 4c). In addition, most of the TF genes showed > 2-fold higher expression in 'Tonghua-3' than in 'Tangwei' at the R stage. In particular, a few members, such as ERF11 (VIT_07s0141g00690), MYB105 (VIT_01s0026g02600), and WRKY70 (VIT_13s0067g03140), showed > 100-fold higher expression in 'Tonghua-3' (Fig. 4d).
    Figure 4.  Differentially expressed transcription factor (TF) genes. (a) The number of differentially expressed genes (DEGs) in different TF families; (b) Number of differentially expressed TF genes, numbers of overlapping differentially expressed TF genes, and (c) categorization of expression fold change (FC) for members of eight TF families between 'Tangwei' and 'Tonghua-3' at green hard (G), véraison (V), and ripe (R) stages; (d) Heatmap expression profiles of the three most strongly differentially expressed TF genes from each of eight TF families.
    We constructed a gene co-expression network using the weighted gene co-expression network analysis (WGCNA) package, which uses a systems biology approach focused on understanding networks rather than individual genes. In the network, 17 distinct modules (hereafter referred to by color as portrayed in Fig. 5a), with module sizes ranging from 91 (antiquewhite4) to 1,917 (magenta) were identified (Supplemental Table S7). Of these, three modules (ivory, orange and blue) were significantly correlated with resveratrol content, cultivar ('Tonghua-3'), and developmental stage (R). The blue module showed the strongest correlation with resveratrol content (cor = 0.6, p-value = 0.008) (Fig. 5b). KEGG enrichment analysis was carried out to further analyze the genes in these three modules. Genes in the ivory module were significantly enriched for phenylalanine metabolism (ko00360), stilbenoid, diarylheptanoid and gingerol biosynthesis (ko00945), and flavonoid biosynthesis (ko00941), whereas the most highly enriched terms of the blue and orange modules were plant-pathogen interaction (ko04626), plant hormone signal transduction (ko04075) and circadian rhythm-plant (ko04712) (Supplemental Fig. S1). Additionally, a total of 36 genes encoding TFs including in 15 ERFs, 10 WRKYs, six bHLHs, two MYBs, one MADs-box, one HSF and one TRY were identified as co-expressed with one or more STSs in these three modules (Fig. 5c and Supplemental Table S8), suggesting that these TFs may participate in the STS regulatory network.
    Figure 5.  Results of weighted gene co-expression network analysis (WGCNA). (a) Hierarchical clustering tree indicating co-expression modules; (b) Module-trait relationship. Each row represents a module eigengene, and each column represents a trait. The corresponding correlation and p-value are indicated within each module. Res, resveratrol; TW, 'Tangwei'; TH, 'Tonghua-3'; (c) Transcription factors and stilbene synthase gene co-expression networks in the orange, blue and ivory modules.
    To assess the reliability of the RNA-seq data, 12 genes determined to be differentially expressed by RNA-seq were randomly selected for analysis of expression via real-time quantitative PCR (RT-qPCR). This set comprised two PALs, two 4CLs, two STSs, two WRKYs, two LACs, one OMT, and MYB14. In general, these RT-qPCR results strongly confirmed the RNA-seq-derived expression patterns during fruit development in the two cultivars. The correlation coefficients between RT-qPCR and RNA-seq were > 0.6, except for LAC (VIT_02s0154g00080) (Fig. 6).
    Figure 6.  Comparison of the expression patterns of 12 randomly selected differentially expressed genes by RT-qPCR (real-time quantitative PCR) and RNA-seq. R-values are correlation coefficients between RT-qPCR and RNA-seq. FPKM, fragments per kilobase of transcript per million fragments mapped; TW, 'Tangwei'; TH, 'Tonghua-3'; G, green hard; V, véraison; R, ripe.
    Grapevines are among the most important horticultural crops worldwide[26], and recently have been the focus of studies on the biosynthesis of resveratrol. Resveratrol content has previously been found to vary depending on cultivar as well as environmental stresses[27]. In a study of 120 grape germplasm cultivars during two consecutive years, the extractable amounts of resveratrol in berry skin were significantly higher in seeded cultivars than in seedless ones, and were higher in both berry skin and seeds in wine grapes relative to table grapes[28]. Moreover, it was reported that total resveratrol content constantly increased from véraison to complete maturity, and ultraviolet-C (UV-C) irradiation significantly stimulated the accumulation of resveratrol of berry during six different development stages in 'Beihong' (V. vinifera × V. amurensis)[9]. Intriguingly, a recent study reported that bud sport could lead to earlier accumulation of trans-resveratrol in the grape berries of 'Summer Black' and its bud sport 'Nantaihutezao' from the véraison to ripe stages[29]. In the present study, resveratrol concentrations in seven accessions were determined by high performance liquid chromatography (HPLC) in the seed, pulp and skin at three developmental stages (G, V and R). Resveratrol content was higher in berry skins than in pulp or seeds, and were higher in the wild Chinese accessions compared with the domesticated cultivars. The highest resveratrol content (2.99 µg g−1 FW) was found in berry skins of 'Tonghua-3' at the R stage (Table 1). This is consistent with a recent study of 50 wild Chinese accessions and 45 cultivars, which reported that resveratrol was significantly higher in berry skins than in leaves[30]. However, we did not detect trans-resveratrol in the skins of 'Tangwei' during the G or V stages (Table 1). To explore the reason for the difference in resveratrol content between 'Tangwei' and 'Tonghua-3', as well as the regulation mechanism of resveratrol synthesis and accumulation during berry development, we used transcriptional profiling to compare gene expression between these two accessions at the G, V, and R stages. After sequence read alignment and transcript assembly, 23,649 and 23,557 unigenes were documented in 'Tangwei' and 'Tonghua-3', respectively. As anticipated, due to the small number of structures sampled, this was less than that (26,346) annotated in the V. vinifera reference genome[18]. Depending on the sample, 80.47%−88.86% of sequence reads aligned to a single genomic location (Supplemental Table S1); this is similar to the alignment rate of 85% observed in a previous study of berry development in Vitis vinifera[19]. Additionally, 1751 novel transcripts were excavated (Supplemental Table S2) after being compared with the V. vinifera reference genome annotation information[18, 31]. A similar result was also reported in a previous study when transcriptome analysis was performed to explore the underlying mechanism of cold stress between Chinese wild Vitis amurensis and Vitis vinifera[32]. We speculate that these novel transcripts are potentially attributable to unfinished V. vinifera reference genome sequence (For example: quality and depth of sequencing) or species-specific difference between Vitis vinifera and other Vitis. In our study, the distribution of genes based on expression level revealed an inverse trend from G, V to R between 'Tangwei' and 'Tonghua-3' (Fig. 1). Furthermore, analysis of DEGs suggested that various cellular processes including metabolic process and catalytic activity were altered between the two cultivars at all three stages (Fig. 2 and Supplemental Table S3S5). These results are consistent with a previous report that a large number of DEGs and 100 functional subcategories were identified in 'Tonghua-3' grape berries after exposure to UV-C radiation[8]. Resveratrol biosynthesis in grapevine is dependent on the function of STSs, which compete with the flavonoid branch in the phenylalanine metabolic pathway. Among the DEGs detected in this investigation, genes directly involved in the resveratrol synthesis pathway, STSs, C4Hs and 4CLs, were expressed to significantly higher levels in 'Tonghua-3' than in 'Tangwei' during V and R. On the other hand, DEGs representing the flavonoid biosynthesis pathway were upregulated in 'Tangwei', but downregulated in 'Tonghua-3' (Fig. 3 and Supplemental Table S6). These expression differences may contribute to the difference in resveratrol content between the two cultivars at these stages. We note that 'Tangwei' and 'Tonghua-3' are from two highly diverged species with different genetic backgrounds. There might be some unknown genetic differences between the two genomes, resulting in more than 60 functional subcategories being enriched (Fig. 2d) and the expression levels of genes with putative roles in resveratrol biosynthesis being significantly higher in 'Tonghua-3' than in 'Tangwei' during V and R (Fig. 3). A previous proteomic study also reported that the expression profiles of several enzymes in the phenylalanine metabolism pathway showed significant differences between V. quinquangularis accession 'Danfeng-2' and V. vinifera cv. 'Cabernet Sauvignon' at the véraison and ripening stages[33]. In addition, genes such as RSGT, OMT and LAC involved in the production of derivatized products of resveratrol were mostly present at the G and V stages of 'Tangwei', potentially resulting in limited resveratrol accumulation. However, we found that most of these also revealed relatively high expression at R in 'Tonghua-3' (Fig. 3). Despite this situation, which does not seem to be conducive for the accumulation of resveratrol, it still showed the highest content (Table 1). It has been reported that overexpression of two grapevine peroxidase VlPRX21 and VlPRX35 genes from Vitis labruscana in Arabidopsis may be involved in regulating stilbene synthesis[34], and a VqBGH40a belonging to β-glycoside hydrolase family 1 in Chinese wild Vitis quinquangularis can hydrolyze trans-piceid to enhance trans-resveratrol content[35]. However, most studies mainly focus on several TFs that participate in regulation of STS gene expression, including ERFs, MYBs and WRKYs[13, 15, 17]. For example, VvWRKY18 activated the transcription of VvSTS1 and VvSTS2 by directly binding the W-box elements within the specific promoters and resulting in the enhancement of stilbene phytoalexin biosynthesis[36]. VqWRKY53 promotes expression of VqSTS32 and VqSTS41 through participation in a transcriptional regulatory complex with the R2R3-MYB TFs VqMYB14 and VqMYB15[37]. VqMYB154 can activate VqSTS9/32/42 expression by directly binding to the L5-box and AC-box motifs in their promoters to improve the accumulation of stilbenes[38]. In this study, we found a total of 757 TF-encoding genes among the DEGs, including representatives of the MYB, AP2/ERF, bHLH, NAC, WRKY, bZIP and HB-HD-ZIP families. The most populous family was MYB, representing 76 DEGs at G, V and R between 'Tangwei' and 'Tonghua-3' (Fig. 4). A recent report indicated that MYB14, MYB15 and MYB13, a third uncharacterized member of Subgroup 2 (S2), could bind to 30 out of 47 STS family genes. Moreover, all three MYBs could also bind to several PAL, C4H and 4CL genes, in addition to shikimate pathway genes, the WRKY03 stilbenoid co-regulator and resveratrol-modifying gene[39]. VqbZIP1 from Vitis quinquangularis has been shown to promote the expression of VqSTS6, VqSTS16 and VqSTS20 by interacting with VqSnRK2.4 and VqSnRK2.6[40]. In the present study, we found that a gene encoding a bZIP-type TF (VIT_12s0034g00110) was down-regulated in 'Tangwei', but up-regulated in 'Tonghua-3', at G, V and R (Fig. 4). We also identified 36 TFs that were co-expressed with 17 STSs using WGCNA analysis, suggesting that these TFs may regulate STS gene expression (Fig. 5 and Supplemental Table S8). Among these, a STS (VIT_16s0100g00880) was together co-expressed with MYB14 (VIT_07s0005g03340) and WRKY24 (VIT_06s0004g07500) that had been identified as regulators of STS gene expression[13, 15]. A previous report also indicated that a bHLH TF (VIT_11s0016g02070) had a high level of co-expression with STSs and MYB14/15[15]. In the current study, six bHLH TFs were identified as being co-expressed with one or more STSs and MYB14 (Fig. 5 and Supplemental Table S8). However, further work needs to be done to determine the potential role of these TFs that could directly target STS genes or indirectly regulate stilbene biosynthesis by formation protein complexes with MYB or others. Taken together, these results identify a small group of TFs that may play important roles in resveratrol biosynthesis in grapevine. In summary, we documented the trans-resveratrol content of seven grapevine accessions by HPLC and performed transcriptional analysis of the grape berry in two accessions with distinct patterns of resveratrol accumulation during berry development. We found that the expression levels of genes with putative roles in resveratrol biosynthesis were significantly higher in 'Tonghua-3' than in 'Tangwei' during V and R, consistent with the difference in resveratrol accumulation between these accessions. Moreover, several genes encoding TFs including MYBs, WRKYs, ERFs, bHLHs and bZIPs were implicated as regulators of resveratrol biosynthesis. The results from this study provide insights into the mechanism of different resveratrol accumulation in various grapevine accessions. V. davidii 'Tangwei', V. amurensis × V. Vinifera 'Beibinghong'; V. amurensis 'Tonghua-3' and 'Shuangyou'; V. vinifera × V. labrusca 'Jumeigui'; V. vinifera 'Red Globe' and 'Thompson Seedless' were maintained in the grapevine germplasm resource at Northwest A&F University, Yangling, Shaanxi, China (34°20' N, 108°24' E). Fruit was collected at the G, V, and R stages, as judged by skin and seed color and soluble solid content. Each biological replicate comprised three fruit clusters randomly chosen from three plants at each stage. About 40−50 representative berries were separated into skin, pulp, and seed, and immediately frozen in liquid nitrogen and stored at −80 °C. Resveratrol extraction was carried out as previously reported[8]. Quantitative analysis of resveratrol content was done using a Waters 600E-2487 HPLC system (Waters Corporation, Milford, MA, USA) equipped with an Agilent ZORBAX SB-C18 column (5 µm, 4.6 × 250 mm). Resveratrol was identified by co-elution with a resveratrol standard, and quantified using a standard curve. Each sample was performed with three biological replicates. Three biological replicates of each stage (G, V and R) from whole berries of 'Tangwei' and 'Tonghua-3' were used for all RNA-Seq experiments. Total RNA was extracted from 18 samples using the E.Z.N.A. Plant RNA Kit (Omega Bio-tek, Norcross, GA, USA). For each sample, sequencing libraries were constructed from 1 μg RNA using the NEBNext UltraTM RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA). The library preparations were sequenced on an Illumina HiSeq2500 platform (Illumina, San Diego, CA, USA) at Biomarker Technologies Co., Ltd. (Beijing, China). Raw sequence reads were filtered to remove low-quality reads, and then mapped to the V. vinifera 12X reference genome[18, 31] using TopHat v.2.1.0[41]. The mapped reads were assembled into transcript models using Stringtie v2.0.4[42]. Transcript abundance and gene expression levels were estimated as FPKM[43]. The formula is as follows:
    FPKM =cDNA FragmentsMapped Fragments(Millions)× Transcript Length (kb)
    Biological replicates were evaluated using Pearson's Correlation Coefficient[44] and principal component analysis. DEGs were identified using the DEGSeq R package v1.12.0[45]. A false discovery rate (FDR) threshold was used to adjust the raw P values for multiple testing[46]. Genes with a fold change of ≥ 2 and FDR < 0.05 were assigned as DEGs. GO and KEGG enrichment analyses of DEGs were performed using GOseq R packages v1.24.0[47] and KOBAS v2.0.12[48], respectively. Co-expression networks were constructed based on FPKM values ≥ 1 and coefficient of variation ≥ 0.5 using the WGCNA R package v1.47[49]. The adjacency matrix was generated with a soft thresholding power of 16. Then, a topological overlap matrix (TOM) was constructed using the adjacency matrix, and the dissimilarity TOM was used to construct the hierarchy dendrogram. Modules containing at least 30 genes were detected and merged using the Dynamic Tree Cut algorithm with a cutoff value of 0.25[50]. The co-expression networks were visualized using Cytoscape v3.7.2[51]. RT-qPCR was carried out using the SYBR Green Kit (Takara Biotechnology, Beijing, China) and the Step OnePlus Real-Time PCR System (Applied Biosystems, Foster, CA, USA). Gene-specific primers were designed using Primer Premier 5.0 software (PREMIER Biosoft International, Palo Alto, CA, USA). Cycling parameters were 95 °C for 30 s, 42 cycles of 95 °C for 5 s, and 60 °C for 30 s. The grapevine ACTIN1 (GenBank Accession no. AY680701) gene was used as an internal control. Each reaction was performed in triplicate for each of the three biological replicates. Relative expression levels of the selected genes were calculated using the 2−ΔΔCᴛ method[52]. Primer sequences are listed in Supplemental Table S9. This research was supported by the National Key Research and Development Program of China (2019YFD1001401) and the National Natural Science Foundation of China (31872071 and U1903107).
  • The authors declare that they have no conflict of interest.
  • Al-Bedak OA, Abdel-Sater MA, Abdel-Latif AMA, Abdel-Wahab D. 2020 – Aspergillus creber and A. keveii, two new records as endophytes from wild medicinal plants in Egypt. Journal of Multidisciplinary Sciences 2, 1–9.

    Google Scholar

    Al-Bedak OA, Sayed RM, Hassan SH. 2019 – A new low-cost method for long-term preservation of filamentous fungi. Biocatalysis and Agricultural Biotechnology 22, 101417. doi: 10.1016/j.bcab.2019.101417

    CrossRef    Google Scholar

    Al-Snafi A. 2015 – The chemical contents and pharmacological effects of Anagallis arvensis-A review. International Journal of Pharmacy 5, 37–41.

    Google Scholar

    Anwar F, Latif S, Ashraf M, Gilani AH. 2007 – Moringa oleifera: a food plant with multiple medicinal uses. Phytotherapy Research: An International Journal Devoted to Pharmacological and Toxicological Evaluation of Natural Product Derivatives 21, 17–25.

    Google Scholar

    Bailey MJ, Biely P, Poutanen K. 1992 – Interlaboratory testing of methods for assay of xylanase activity. Journal of biotechnology 23, 257–270. doi: 10.1016/0168-1656(92)90074-J

    CrossRef    Google Scholar

    Booth C. 1971 – The genus Fusarium. Commonwealth Mycological Institute. Kew, Surrey 237 p.

    Google Scholar

    Carbungco E, Pedroche N, Panes V, De la Cruz T. 2015 – Identification and characterization of endophytic fungi associated with the leaves of Moringa oleifera Lam, I International Symposium on Moringa 1158, 373–380.

    Google Scholar

    Carroll G, Petrini O. 1983 – Patterns of substrate utilization by some fungal endophytes from coniferous foliage. Mycologia 75, 53–63. doi: 10.1080/00275514.1983.12021637

    CrossRef    Google Scholar

    Choi Y, Hodgkiss I, Hyde K. 2005 – Enzyme production by endophytes of Brucea javanica. J Agric Technol 1, 55–66.

    Google Scholar

    De Aldana BRV, Bills G, Zabalgogeazcoa I. 2013 – Are endophytes an important link between airborne spores and allergen exposure? Fungal Diversity 60, 33–42. doi: 10.1007/s13225-013-0223-z

    CrossRef    Google Scholar

    de Hoog GS, Guarro J, Gené J, Figueras M. 2000 – Atlas of clinical fungi. Centraalbureau voor Schimmelcultures (CBS) 1126 p.

    Google Scholar

    Domsch K, Gams W, Anderson T. 2007– Compendium of soil fungi. 1–672. IHW-Verlag, Eching, Germany 672 p.

    Google Scholar

    Edor SP, Edogbanya OP, Kutshik JR. 2018 – Cellulase activity of Aspergillus niger in the biodegradation of rice husk. MOJ Biology and Medicine 3(3), 49–51

    Google Scholar

    Ellis M. 1976 – More Dematiaceous Hyphomycetes., (Commonwealth Mycological Institute: Kew, Surrey) 507 p.

    Google Scholar

    Gessner R. 1980 – Degradative enzyme production by salt-marsh fungi. Botanica Marina 23, 133–139.

    Google Scholar

    Gherbawy Y, Gashgari R. 2014 – Molecular characterization of fungal endophytes from Calotropis procera plants in Taif region (Saudi Arabia) and their antifungal activities. Plant Biosystems-An International Journal Dealing with all Aspects of Plant Biology 148, 1085–1092. doi: 10.1080/11263504.2013.819043

    CrossRef    Google Scholar

    Hamayun M, Khan SA, Khan AL, Rehman G et al. 2010 – Gibberellin production and plant growth promotion from pure cultures of Cladosporium sp. MH-6 isolated from cucumber (Cucumis sativus L. ). Mycologia 102, 989–995. doi: 10.3852/09-261

    CrossRef    Google Scholar

    Ismail MA, Moubasher AH, Mohamed RA, Al-Bedak OA. 2017 – Extremophilic fungi and chemical analysis of hypersaline, alkaline lakes of Wadi-El-Natrun, Egypt. International Journal of Technical Research and Science 1, 345–363.

    Google Scholar

    Ismail MA, Moubasher AH, Mohamed RA, Al-Beddak OA. 2018 – Agro–industrial residues as alternative sources for cellulases and xylanases production and purification of xylanase produced by Aspergillus flavus AUMC 10331 isolated from extreme habitat. Current Research in Environmental & Applied Mycology 8, 313–322.

    Google Scholar

    Jalis H, Ahmad A, Khan S, Sohail M. 2014 – Utilization of apple peels for the production of plant cell-wall degrading enzymes by Aspergillus fumigatus MS16. J Anim Plant Sci 24, 64–67.

    Google Scholar

    Kaur M, Kalia A. 2012 – Convolvulus arvensis: A useful weed. Int J Pharm Pharm Sci 4, 38–40.

    Google Scholar

    KC S, Upadhyaya J, Joshi DR, Lekhak B et al. 2020 – Production, Characterization, and Industrial Application of Pectinase Enzyme Isolated from Fungal Strains. Fermentation 6(59), 2–10.

    Google Scholar

    Khairnar AK, Bhamare S, Bhamare H. 2012 – Calotropis procera: an ethnopharmacological update. Advance Research in Pharmaceuticals and Biologicals 2, 142–156.

    Google Scholar

    Khan AL, Waqas M, Hussain J, Al-Harrasi A, Lee I-J. 2014 – Fungal endophyte Penicillium janthinellum LK5 can reduce cadmium toxicity in Solanum lycopersicum (Sitiens and Rhe). Biology and fertility of soils 50, 75–85. doi: 10.1007/s00374-013-0833-3

    CrossRef    Google Scholar

    Khan Marwat S, Khan EA, Baloch MS, Sadiq M et al. 2017 – Ricinus cmmunis: Ethnomedicinal uses and pharmacological activities. Pakistan journal of pharmaceutical sciences 30(5), 1815–1827.

    Google Scholar

    Khan R, Shahzad S, Choudhary MI, Khan SA, Ahmad A. 2007 – Biodiversity of the endophytic fungi isolated from Calotropis procera (Ait. ) R. Br. Pakistan Journal of Botany 39, e2239.

    Google Scholar

    Krishnamurthy YL, Naik SB, Jayaram S. 2008 – Fungal communities in herbaceous medicinal plants from the Malnad region, Southern India. Microbes and environments 23, 24–28. doi: 10.1264/jsme2.23.24

    CrossRef    Google Scholar

    Kuhad RC, Gupta R, Singh A. 2011 – Microbial cellulases and their industrial applications. Enzyme research 2011, 1–10.

    Google Scholar

    Leslie J, Summerell B. 2006 – The Fusarium Laboratory Manual Blackwell Publishing. Ames, Iowa 382 p.

    Google Scholar

    Li J-X, Zhang F, Jiang D-D, Li J et al. 2020 – Diversity of Cellulase-Producing Filamentous Fungi From Tibet and Transcriptomic Analysis of a Superior Cellulase Producer Trichoderma harzianum LZ117. Frontiers in Microbiology 11, 1617. doi: 10.3389/fmicb.2020.01617

    CrossRef    Google Scholar

    Miller GL. 1959 – Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical chemistry 31, 426–428. doi: 10.1021/ac60147a030

    CrossRef    Google Scholar

    Moubasher AH. 1993 – Soil fungi in Qatar and other Arab countries. The Centre for Scientific and Applied Research, University of Qatar 568 p.

    Google Scholar

    Moubasher AH, Ismail MA, Mohamed RA, Al-Bedak OA. 2016 – Xylanase and cellulase production under extreme conditions in submerged fermentation by some fungi isolated from hypersaline, alkaline lakes of Wadi-El-Natrun, Egypt. Journal of Basic & Applied Mycology (Egypt) 7, 19–32.

    Google Scholar

    Muhammad G, Hussain MA, Anwar F, Ashraf M, Gilani AH. 2015 – Alhagi: a plant genus rich in bioactives for pharmaceuticals. Phytotherapy research 29, 1–13. doi: 10.1002/ptr.5222

    CrossRef    Google Scholar

    Oumer OJ. 2017 – Pectinase: substrate, production and their biotechnological applications. International Journal of Environment, Agriculture and Biotechnology 2, 238761.

    Google Scholar

    Oumer OJ, Abate D. 2018 – Screening and molecular identification of pectinase producing microbes from coffee pulp. BioMed research international 2018, 1–7.

    Google Scholar

    Patil MG, Pagare J, Patil SN, Sidhu AK. 2015 – Extracellular enzymatic activities of endophytic fungi isolated from various medicinal plants. Int J Curr Microbiol App Sci 4, 1035–1042.

    Google Scholar

    Petrini O. 1991 – Fungal Endophytes of Tree Leaves. In: Andrews J.H., Hirano S.S. (eds) Microbial Ecology of Leaves. Brock/Springer Series in Contemporary Bioscience. Springer, New York, NY. 179–197.

    Google Scholar

    Pitt JI. 1979 – The genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces.

    Google Scholar

    Prabavathy D, Valli NC. 2012 – Screening for extracellular enzymes and production of cellulase by an endophytic Aspergillus sp., using cauliflower stalk as substrate. International Journal on Applied Bioengineering 6(2), 40–45.

    Google Scholar

    Raviraja N. 2005 – Fungal endophytes in five medicinal plant species from Kudremukh Range, Western Ghats of India. Journal of Basic Microbiology: An International Journal on Biochemistry, Physiology, Genetics, Morphology, and Ecology of Microorganisms 45, 230–235.

    Google Scholar

    Rodriguez R, White Jr J, Arnold AE, Redman aRa. 2009 – Fungal endophytes: diversity and functional roles. New phytologist 182, 314–330. doi: 10.1111/j.1469-8137.2009.02773.x

    CrossRef    Google Scholar

    Samson R, Hoekstra E, Frisvad J, Filtenborg O. 2004 – Introduction to food and airborne fungi 7th ed. Utrecht: Centraal Bureau voor Schimmelcultures 389 p.

    Google Scholar

    Schardl CL, Leuchtmann A, Spiering MJ. 2004 – Symbioses of grasses with seedborne fungal endophytes. Annu. Rev. Plant Biol. 55, 315–340. doi: 10.1146/annurev.arplant.55.031903.141735

    CrossRef    Google Scholar

    Shubha J, Srinivas C. 2017 – Diversity and extracellular enzymes of endophytic fungi associated with Cymbidium aloifolium L. African Journal of Biotechnology 16, 2248–2258.

    Google Scholar

    Simmons EG. 2007 – Alternaria: An Indentification Manual. Utrecht, The Netherlands: CBS Fungal Biodiversity Centre. 775 p.

    Google Scholar

    Singh LP, Gill SS, Tuteja N. 2011 – Unraveling the role of fungal symbionts in plant abiotic stress tolerance. Plant signaling & behavior 6, 175–191.

    Google Scholar

    Sopalun K, Iamtham S. 2020 – Isolation and screening of extracellular enzymatic activity of endophytic fungi isolated from Thai orchids. South African Journal of Botany (2020), 1–7.

    Google Scholar

    Strobel G, Daisy B, Castillo U, Harper J. 2004 – Natural products from endophytic microorganisms. Journal of Natural products 67, 257–268. doi: 10.1021/np030397v

    CrossRef    Google Scholar

    Sunitha V, Nirmala Devi D, Srinivas C. 2013 – Extracellular enzymatic activity of endophytic fungal strains isolated from medicinal plants. World Journal of Agricultural Sciences 9, 1–9.

    Google Scholar

    Syed S, Fatima N, Kabeer G. 2016 – Portulaca oleracea L. : a mini review on phytochemistry and pharmacology. International journal of Biology and Biotechnology 13, 637–641.

    Google Scholar

    Xue Y, Han J, Li Y, Liu J et al. 2020 – Promoting cellulase and hemicellulase production from Trichoderma orientalis EU7-22 by overexpression of transcription factors Xyr1 and Ace3. Bioresource Technology 296, 122355. doi: 10.1016/j.biortech.2019.122355

    CrossRef    Google Scholar

    Zou W, Meng J, Lu H, Chen G et al. 2000 – Metabolites of Colletotrichum gloeosporioides, an endophytic fungus in Artemisia mongolica. Journal of Natural Products 63, 1529–1530. doi: 10.1021/np000204t

    CrossRef    Google Scholar

  • Cite this article

    MA Abdel-Sater, AMA Abdel-Latif, DA Abdel-Wahab, OA Al-Bedak. 2021. Endophytic mycobiota of wild medicinal plants from New Valley Governorate, Egypt and quantitative assessment of their cell wall degrading enzymes. Studies in Fungi 6(1):78−91 doi: 10.5943/sif/6/1/4
    MA Abdel-Sater, AMA Abdel-Latif, DA Abdel-Wahab, OA Al-Bedak. 2021. Endophytic mycobiota of wild medicinal plants from New Valley Governorate, Egypt and quantitative assessment of their cell wall degrading enzymes. Studies in Fungi 6(1):78−91 doi: 10.5943/sif/6/1/4

Figures(2)  /  Tables(5)

Article Metrics

Article views(3808) PDF downloads(590)

ARTICLE   Open Access    

Endophytic mycobiota of wild medicinal plants from New Valley Governorate, Egypt and quantitative assessment of their cell wall degrading enzymes

Studies in Fungi  6 Article number: 4  (2021)  |  Cite this article

Abstract: The present study isolated and identified 32 species of endophytic mycobiota belonging to 18 genera associated with 8 wild medicinal plants collected from El-Kharga Oasis, New Valley Governorate, Egypt. Fusarium was the most common genus followed by Alternaria and Aspergillus. Convolvulus arvensis was the plant with the highest number of endophytes over the other plant species, while Moringa oleifera reported the lowest number of endophytes. In addition, the entomopathogenic fungus Beauveria bassiana; was recorded for the first time from leaves of Portulaca oleracea. One hundred and twenty-three isolates representing 32 species were screened for their abilities to produce pectinase, carboxy methyl cellulase (CMCase) and avicellase enzymes on sucrose free-Cz supplemented, individually with 1% pectin or 1% CMC or 1% avicel as a sole carbon source, respectively. Ninety-four isolates produced pectinase while 66 isolates produced cellulases. The quantitative assays of the three enzymes for high-producers were performed in submerged fermentation using sucrose-free Cz broth. Aspergillus was the superior in the production of the three enzymes with the potent strains were A. terreus AUMC 14287 for CMCase (22.0 IU/ml/min) and avicellase (47.868 IU/ml/min) and A. terreus AUMC 14278 for pectinase (225.43 IU/ml/min).

  • Endophytic microorganisms colonize in plant tissues in which they spend part or all their life cycle without causing disease symptoms in the host (Petrini 1991). Fungal endophytes may inhabit in different organs of the host including leaves, stems, bark, roots, fruits, flowers and seeds (Rodriguez et al. 2009). Generally, in this symbiotic relationship fungal endophytes receive shelter and nutrients from the host, while the host plant may benefit from an array of attributes which include protection against natural enemies such as pathogens and herbivores (Schardl et al. 2004, Singh et al. 2011), plant growth promotion (Hamayun et al. 2010) and increasing the resistance of plants to abiotic stresses such as salinity and heavy metal toxicity in soil (Khan et al. 2014). Some medicinal plants are known for harboring endophytic fungi, which are important sources of various bioactive secondary metabolites and enzymes valuable for the pharmaceutical industry (Zou et al. 2000, Strobel et al. 2004, Krishnamurthy et al. 2008).

    Endophytic fungi are relatively unexplored producers of metabolites useful in pharmaceutical and agricultural industries. A single endophyte can produce several bioactive metabolites. As a result, the role of endophytes in the production of various natural products with greater bioactivity have received increased attention (Prabavathy & Valli 2012). Pectinases and cellulases, besides other enzymes, are the most important enzymes produced by endophytic fungi as a resistance mechanism against pathogenic invasion and to obtain nutrition from the host. These enzymes have various industrial applications, thus of major interest. Increasing efforts are being taken to characterize and identify endophytic fungi from medicinal plants. Therefore, the present work was designed to study the biodiversity of endophytic fungi in some wild medicinal plants from the New Valley Governorate, Egypt, and to evaluate their ability to produce extracellular pectinases and cellulases.

  • The New Valley Governorate is located at the Western Desert of Egypt. It encompasses 440, 098 km2, which is approximately 44% of the total area of Egypt and 66% of the area of Western Sahara. It is demarcated by the Governorates of Minya, Assiut, Sohag, Qena and Aswan from the east, by Libya and the Governorates of Matrouh and the Marine Oasis of the 6th of October City from the West and by Sudan from the South. The New Valley includes four large Oases namely El-Kharga (the sampling sites), El-Dakhla, El-Bahariya and El-Farafra, and the capital is El-Kharga (Fig. 1).

    Figure 1.  Location of the New Valley Governorate showing study site.

  • Healthy and mature plant leaves and roots of eight wild medicinal plants were collected from El-Kharga Oasis, New Valley Governorate once during April 2018. Ten replicates from each of Alhagi graecorum, Anagallis arvensis, Calotropis procera, Chenopodium ambrosioides, Convolvulus arvensis, Moringa oleifera, Portulaca oleracea, and Ricinus communis plants were collected in sterile polyethylene bags and promptly brought to the laboratory for isolation of fungi. The plant species collected in the current investigation were identified according to morphological features and taxonomical characters at the Assiut University Herbarium, Department of Botany and Microbiology, Faculty of Science, Assiut University, Assiut, Egypt (Fig. 2).

    Figure 2.  Wild medicinal plant species collected from El-Kharga Oasis, the New Valley Governorate, Egypt, during April 2018.

  • Prior to surface sterilization, leaves and roots of each sample were thoroughly washed with tap water to remove the dust followed by distilled water. The samples were then cut into 5-cm segments. The samples were surface sterilized using the following sequence; 5% sodium hypochlorite for 3 min, 70% ethanol for 1 min, and washing with sterile distilled water 3 times each for 1 min. In aseptic conditions, both ends of each segment (1 cm) was cut off to produce a 3-cm segments (Al-Bedak et al. 2020).

  • Segments of each sample were plated on Petri-dishes containing 1% glucose-Cz with the following composition (g/l): Glucose, 10; Na2NO3, 2; K2HPO4, 1; KCl, 0.5; MgSO4.7H2O, 0.5; FeSO4, 0.01; ZnSO4, 0.01; CuSO4, 0.005; Rose Bengal, 0.05; chloramphenicol, 0.25; agar, 15 and the final pH 7.3 (Ismail et al. 2017). The plates were incubated for 7-21 days at 25℃. Counts of CFUs of each fungal isolate were calculated per 25 segments in every sample. The obtained fungi were identified morphologically to the species level at the Assiut University Mycological Centre according to their macroscopic and microscopic characteristics. Pure cultures of the fungal strains were preserved for further investigations on PDA slants, as well as on cotton balls (Al-Bedak et al. 2019) at 4℃ in the culture collection of the Assiut University Mycological Centre.

  • The obtained fungi in this study were identified morphologically to the species level at the Assiut University Mycological Centre according to their macroscopic and microscopic characteristics. The following references were used for the identification of fungal genera and species (purely morphologically, based on macroscopic and microscopic features): Booth (1971), Ellis (1976), Pitt (1979), Domsch et al. (2007), Moubasher (1993), de Hoog et al. (2000), Samson et al. (2004), Leslie & Summerell (2006), Simmons (2007) and Al-Bedak et al. (2020).

  • Production of pectinase and endoglucanase was detected on sucrose-free Czapek's agar medium amended with pectin (from citrus peel) and CMC as a sole carbon source, respectively. 50 µl of spore suspension from 7-day-old culture of each fungal strain was individually added to each 5-mm diameter well on the agar plate (Moubasher et al. 2016). The inoculated plates were incubated for 2 days at 30ºC. The clear zones formed around the wells were more visible when the plates were flooded with 0.25% (w/v) aqueous iodine solution. The diameters of the clear zones were measured (in mm) against the brown color of the test medium indicating enzyme production.

  • All positive fungal strains were grown, individually in 250-ml Erlenmeyer conical flasks each containing 50 ml sucrose-free Czapek's broth medium supplemented with 1% pectin or 1% CMC as sole carbon source. The flasks were then inoculated individually with 1 ml spore suspension containing 1 x 107 spore/ml of 7-day-old culture of the tested strains. The inoculated flasks were then incubated at 30ºC in shaking condition of 150 rpm for 7 days.

  • After incubation period, the flasks contents were individually filtered through filter papers (Whatman No. 1) and the filtrate was then centrifuged at 10000 xg for 10 min at 4ºC. The clear supernatants were used as a source for CMCase or pectinase enzyme.

  • The enzyme production was determined by mixing 0.9 ml of 1% pectin (prepared in 50 mM Na-citrate buffer, pH 5.0) with 0.1 ml of filtered crude enzyme, and the mixture was incubated at 50℃ for 15 min in a water bath (Bailey et al. 1992). The reaction was stopped by the addition of 2 ml of 3, 5-dinitrosalicylic acid (DNS) and the contents were boiled in water bath for 10 min (Miller 1959). After cooling, absorbance was measured at 540 nm using Cary 60 UV-Vis spectrophotometer. The amount of reducing sugar liberated was quantified using calibration curve of glucose. One unit of pectinase is defined as the amount of enzyme that liberates 1 µmol of glucose equivalents per minute under the standard assay conditions.

  • The cellulases activity was determined by mixing 0.9 ml of 1% CMC or 1% avicel (prepared in 50 mM Na-citrate buffer, pH 5.0) with 0.1 ml of filtered crude enzyme, and the mixture was incubated at 50℃ for 15 min in a water bath (Bailey et al. 1992). The reaction was stopped by the addition of 2 ml of 3, 5-dinitrosalicylic acid (DNS) and the contents were boiled in water bath for 10 min (Miller 1959). After cooling, absorbance of the developed color was measured at 540 nm using Cary 60 UV-Vis spectrophotometer. The amount of reducing sugar liberated was quantified using calibration curve of glucose. One unit of CMCase or avicellase is defined as the amount of enzyme that liberates 1 µmol of glucose equivalents per minute under the standard assay conditions. Glucose concentration was calculated using the calibration curve.

    Glucoseconcentration= Absorbance  slope (=1.0472)mg/ml(=g/L)Enzymeconcentration= Glucose concentration (g/L)0.00018IU/L

    The enzyme activity (pectinase or CMCase or avicellase) was calculated according to the following formula (Moubasher et al. 2016)

    Enzymeactivity=AbsorbancexDFx(1x)(1y)(1t)(1slope)

    Where: DF = the dilution factor for enzyme, x = the volume of enzyme used, y = the volume of hydrolysate used for assay of reducing sugars, t = the time of hydrolysis, slope is determined from a standard curve

  • A total of 32 species related to 18 genera of endophytic mycobiota were recovered on 1% glucose-Cz at 25℃ from healthy and mature plant leaves and roots of eight wild medicinal plants, collected from El-Kharga Oasis, the New Valley Governorate. The high incidence in genera were recorded in Alternaria, Aspergillus and Fusarium. Fusarium (represented by 2 species) was the most common and encountered total CFU constituting 37.0% of total fungi. It was recovered from 7 plants out of 8. F. oxysporum was the most prevalent species encountering 23.1% of total fungi, however it was recorded from 3 plants only, followed by F. solani giving rise to 13.9% of total fungi and it was the most frequent recovered from 6 plants. Alternaria (7 species) came next to Fusarium and it was comprised 24.0% of total fungi with A. alternata being the most common Alternaria species recorded from 4 plants and was comprised 10.33% of total fungi followed by A. tenuissima (from 4 plants) comprising 7.42% of total fungi. Aspergillus (5 species in addition to 2 unknown species) was the runner of Alternaria comprising 17.5% of total fungi. It was the most frequent genus isolated from all the studied plants. The most prevalent Aspergillus species were A. terreus followed by A. flavus constituting 7.42% and 5.84% of total fungi respectively (Table 1).

    Table 1.  CFUs (calculated to the total CFUs of each fungus per 25 segments of leaves (L) or roots (R) of each plant sample), Gross total CFUs and % gross total CFUs of fungi isolated from 8 wild medicinal plants collected from El-Kharga Oasis, New Valley Governorate on 1 % glucose-Cz at 25℃ during April 2018.

    Fungal genera & species Plant species Gross total
    Alhagi graecorum Convolvulus arvensis Chenopodium ambrosioides Calotropis procera Ricinus communis Angallis arvensis Moringa oleifera Portulaca oleracea
    L R L R L R L R L R L R L R L R CFU %CFU
    Acremonium 4 4 0.89
    A. rutilum 1 1 0.22
    A. sclerotigenum 3 3 0.67
    Alternaria 1 34 36 1 20 15 107 24
    A. alternata 1 21 20 4 46 10.33
    A. brassicicola 1 1 0.22
    A. chlamydospora 8 8 1.79
    A. citri 1 7 8 1.79
    A. citri macularis 4 4 0.89
    A. longipes 3 3 1 7 1.57
    A. tenuissima 10 8 9 6 33 7.42
    Aspergillus 1 10 3 2 1 1 20 10 21 2 2 2 1 2 78 17.5
    A. creber 2 2 0.44
    A. flavus 1 1 1 19 2 1 1 26 5.84
    A. fumigatus 1 1 2 0.44
    A. keveii 1 1 0.22
    A. parasiticus 1 8 1 1 1 12 2.70
    A. terreus 1 9 1 20 1 1 33 7.42
    A. tubingensis 2 2 0.44
    Beauveria bassiana 1 1 0.22
    Chaetomium senegalense 1 1 0.22
    Cladosporium exile 1 1 0.22
    Clonostachys solani 1 1 0.22
    Curvularia spicifera 19 1 20 4.49
    Fusarium 7 33 37 14 12 14 22 6 20 165 37
    F. oxysporum 6 33 36 22 6 103 23.1
    F. solani 1 1 14 12 14 20 62 13.9
    Macrophomina phaseolina 1 4 1 1 7 1.57
    Penicillium olsonii 21 21 4.72
    Pseudoallescheria boydii 2 2 0.44
    Rhizoctonia solani 1 2 3 0.66
    Rhizopus microspores 1 1 0.22
    Sarocladium kiliense 2 2 0.44
    Scopulariopsis fimicola 1 1 0.22
    Stemphylium botryosum 6 1 6 13 2.92
    Verticillium fungicola 2 2 0.44
    Yeast spp. 1 1 1 1 1 1 8 1 15 3.37
    CFUs 1 37 43 44 36 20 62 23 45 21 52 9 18 0 10 24 445 100
    No. of genera 1 4 5 6 3 4 3 3 5 6 6 3 3 0 2 4
    No. of species 1 6 7 8 5 4 7 4 7 6 9 3 6 0 2 5
    Total CFUs 38 87 56 85 66 61 18 34 445
    Total genera (18) 4 9 6 5 10 7 3 5
    Total species (32) 6 14 9 11 12 10 6 6

    Aspergillus parasiticus, Macrophomina phaseolina were found in 4 plant species, A. longipes and Stemphylium botryosum in 3 plants, A. citri, A. fumigatus, and C. spicifera in 2 plants while Acremonium rutilum, Beauveria bassiana, Chaetomium senegalense, Cladosporium exile, Clonostachys rosea, C. solani, Pseudoallescheria boydii, Rhizoctonia solani, Rhizopus microsporus, Scopulariopsis fimicola, Stemphylium botryosum and Verticillium fungicola were recorded each in one plant species. Convolvulus arvensis was the richest plant with endophytes containing 14 species belonged to 8 genera and recording the highest CFUs of 79 per 25 segments over the remaining plant species, while Moringa oleifera was the poorest in endophytes with 5 species belonging to 2 genera and the lowest CFUs of 13 per 25 segments. It is worth mentioning that Beauveria bassiana; the known entomopathogenic fungus was recorded for the first time from leaves of Portulaca oleracea as an endophyte (Table 1).

  • One-hundred and twenty fungal isolates representing 31 species related to 17 genera of endophytic fungi were screened for their abilities to produce pectinase and endoglucanase on sucrose free-Cz supplemented with 1% pectin or 1% CMC as a sole carbon source, respectively. Ninety-four isolates could produce pectinase enzyme, of which 18 were high producers, 25 moderate and 51 low. 66 isolates could produce cellulase, of which 13 were high producers, 16 moderate and 37 low (Appendix 1).

    Table Appendix 1.  Preliminary screening of pectinases and cellulases production by endophytic fungi recovered from leaves and roots of eight wild medicinal plants collected from El-Kharga Oasis, the New Valley Governorate, Egypt, during April 2018.

    Fungal species Number of isolates tested Preliminary screening
    Pectinases Cellulases
    Positive L M H Positive L M H
    Acremonium 2 2 1 1 2 1 1
    Acremonium rutilum 1 1 1 1 1
    Acremonium sclerotigenum 1 1 1 1 1
    Alternaria 26 20 14 6 6 4 2
    A. alternata 8 7 5 2 2 2
    A. brassicicola 1 1 1
    A. chlamydospora 1 1 1 1 1
    A. citri 3 2 2
    A. citri macularis 2 2 2
    A. longipes 3 3 1 2 1 1
    A. tenuissima 8 6 6
    Aspergillus 35 27 11 9 7 21 6 7 8
    A. flavus 9 7 3 2 2 4 1 2 1
    A. fumigatus 2 1 1 2 1 1
    A. parasiticus 6 3 2 1 2 2
    A. terreus 15 14 5 5 4 12 4 2 6
    A. tubingensis 1
    Aspergillus AY-1 1 1 1 1 1
    Aspergillus AY-2 1 1 1
    Beauveria bassiana 1 1 1
    Chaetomium senegalense 1 1 1
    Cladosporium exile 1 1 1 1 1
    Clonostachys solani 1 1 1 1 1
    Curvularia spicifera 2 1 1 2 2
    Fusarium 23 20 14 3 3 16 13 1 2
    F. oxysporum 13 11 8 2 1 11 10 1
    F. solani 10 9 6 1 2 5 3 1 1
    Macrophomina phaseolina 7 3 3 3 2 1
    Penicillium olsonii 1 1 1 1 1
    Pseudo allescheria boydii 1 1 1 1 1
    Rhizopus microsporus 1 1 1 1 1
    Sarocladium kiliense 1 1 1 1 1
    Scopulariopsis fimicola 1
    Stemphylium botryosum 5 5 3 2 3 3
    Verticillium fungicola 1 1 1 1 1
    Yeast spp. 10 7 3 1 3 6 4 2
    Total isolates 120 94 51 25 18 66 37 16 13
    No. of genera 17 16 10 8 8 14 9 6 5
    No. of species 31 28 17 15 9 23 14 9 8
    Note: H = high producers: ≥ 20 mm, M = moderate: 11-19 mm, L = < 11 mm
  • The quantitative assay of pectinase, CMCase and avicellase for high-producing isolates were performed in submerged fermentation using sucrose-free Cz broth medium amended with 1% pectin or CMC or avicel as the sole carbon source. Of these, 17 isolates could produce pectinase enzyme with a relative activity ranged from 147.84 IU/ml/min to 225.43 IU/ml/min while 14 could produce CMCase (1.84 IU/ml/min – 22.0 IU/ml/min) and avicellase (26.0 IU/ml/min – 47.87 IU/ml/min). Six isolates were found to have the abilities to produce the three enzymes, of which Aspergillus was the superior with the potent strains were A. terreus AUMC 14278 for pectinase activity giving 225.43 IU/ml/min and A. terreus AUMC 14287 for CMCase producing 22.0 IU/ml/min and avicellase recording 47.868 IU/ml/min (Tables 2-4).

    Table 2.  Pectinase production and activity of some endophytic fungi.

    Fungal species AUMC no. Pectinase
    Glucose g/l Production IU/ml Activity IU/ml/min
    Aspergillus flavus 14274 19.27 107.063 147.8
    A. flavus 14289 27.82 154.555 213.4
    A. fumigatus 14283 27.44 152.438 210.5
    A. terreus* 14278 29.4 163.244 225.4
    A. terreus 14287 26.34 146.326 202.0
    A. terreus 14293 26.0 144.814 200.0
    A. terreus 14279 24.14 134.103 185.2
    Cladosporium exile 14294 23.91 132.846 183.4
    Curvularia spicifera 14276 23.6 131.016 180.9
    C. spicifera 14273 29.0 161.255 222.7
    Fusarium solani 14277 23.3 129.472 178.8
    F. solani 14292 21.6 119.954 165.6
    Macrophomina phaseolina 14272 23.253 129.185 178.4
    M. phaseolina 14275 23.0 127.854 176.5
    Penicillium olsonii 14295 23.73 131.843 182.0
    Yeast sp. 14289 24.85 138.066 190.7
    Yeast sp. 14281 22.9 127.275 175.8
    * The highest producer showed in bold

    Table 3.  Endoglucanase (CMCase) production and activity of some endophytic fungi.

    Fungal species AUMC no. Endoglucanase (CMCase)
    Glucose g/l Production IU/ml Activity IU/ml/min
    Aspergillus flavus 14274 0.41 2.282 3.15
    A. fumigatus 14283 0.3 1.684 2.32
    A. terreus 14278 1.9 10.623 14.7
    A. terreus* 14287 2.874 15.966 22.0
    A. terreus 14280 2.68 14.895 20.6
    A. terreus 14282 1.47 8.164 11.3
    A. terreus 14284 1.9 10.579 14.6
    A. terreus 14285 1.5 8.328 11.5
    A. terreus 14288 1.0 5.575 7.7
    Clonostachys rosea 14291 0.24 1.332 1.84
    Curvularia spicifera 14276 1.3 7.167 9.9
    C. spicifera 14273 1.39 7.711 10.65
    Fusarium oxysporum 14290 0.757 4.206 5.8
    F. solani 14286 0.7 3.910 5.4
    * The highest producer showed in bold

    Table 4.  Avicellase production and activity of some endophytic fungi.

    Fungal species AUMC no. Avicellase
    Glucose g/l Production IU/ml Activity IU/ml/min
    Aspergillus flavus 14274 4.213 23.405.5 35.55
    A. fumigatus 14283 4.232 23.512 35.71
    A. terreus 14278 4.049 22.496 34.17
    A. terreus* 14287 5.672 31.51 47.87
    A. terreus 14280 4.418 24.51 37.23
    A. terreus 14282 3.706 20.588 31.3
    A. terreus 14284 3.671 20.395 30.98
    A. terreus 14285 3.876 21.53 32.71
    A. terreus 14288 4.907 27.258 41.41
    Clonostachys rosea 14291 3.466 19.258 29.25
    Curvularia spicifera 14276 3.592 19.958 30.32
    C. spicifera 14273 3.782 21.008 31.91
    Fusarium oxysporum 14290 3.085 17.139 26.0
    F. solani 14286 3.709 20605 31.3
    * The highest producer showed in bold
  • In the current study, endophytic mycobiota in healthy and mature leaves and roots of eight wild medicinal plants were isolated on 1% glucose-Cz at 25℃ from sample collected once in April 2018. This study is considered as the first in the New Valley Governorate, Egypt for evaluation of endophytic fungi from these medicinal plants. There is a growing body of literatures that recognize the importance of endophytic fungi across a number of disciplines in recent years as biological sources of a wide range of valuable compounds including plant growth regulatory, antibacterial, antifungal, antiviral, insecticidal substances to enhance the growth and competitiveness of the host in nature (Anwar et al. 2007, Kaur & Kalia 2012, Khairnar et al. 2012, Al-Snafi 2015, Muhammad et al. 2015, Syed et al. 2016, Khan Marwat et al. 2017).

    The current results revealed that endophytic fungal assemblages were obtained from all plant species examined and some plants were occupying by the same fungal genera and species, indicating that endophytic fungi can be the same in plants belonging to different families. Altogether, 32 species related to 18 genera were recovered from the leaves and roots of all tested plants.

    The high occurrence genera where described by Fusarium, Aspergillus and Alternaria. Fusarium was the most widespread genus retrieved from 7 plants. F. oxysporum is the most dominant led by F. solani. Such latest observations have, to some degree, been compatible with the reports of Raviraja (2005) who researched endophytic fungi in five Brazilian medicinal plants and found that Aspergillus and Penicillium were isolated at high frequencies, however, Fusarium oxysporum was reported at low levels from leaves of two plants tested. Previous studies on plants of the same size as ours have previously been conducted with the genera Fusarium, Aspergillus, Nigrospora, Stachybotrys, Rhizoctonia and Macrophomina from Moringa leaves (Carbungco et al. 2015). Almost similar results were obtained in other studies on Calotropis procera in Karachi (Khan et al. 2007) or in Saudi Arabia (Gherbawy & Gashgari 2014).

    Endophytic fungi produce enzymes such as amylases, cellulase, lipases and proteases, as part of their mechanism to overcome the defense of the host against microbial invasion and to obtain nutrients for their development (Patil et al. 2015). In addition, these enzymes are essential for endophytic fungi to colonize in the plant tissue (Sunitha et al. 2013). The array of enzymes produced differs between fungi and often depends on the host and their ecological factors (Sunitha et al. 2013). In the current study, 120 fungal isolates were screened for their ability to produce pectinase and cellulase. The results obtained revealed that 78.0% of the total isolates tested could produce pectinase enzyme and 55.0% could produce cellulase enzyme.

    The quantitative assay of the three enzymes for high-producers were performed in submerged fermentation using sucrose-free Cz broth. Aspergillus was superior in the production of the three enzymes with the potent strains were A. terreus AUMC 14287 for CMCase and avicellase, and A. terreus AUMC 14278 for pectinase. Almost similar results were reported by (Sunitha et al. 2013) who found that 62.0% and 32.0% of their tested endophytic isolates were positive for pectinase and cellulase respectively, however, their tested fungi were isolated from plants differ from ours. In another study of cellulase activity of fungi inhabiting salt marshes, 100% of the tested isolates showed cellulolytic activity (Gessner 1980), while 66.0 % of fungi isolated from Brucea javanica could produce cellulase enzyme (Choi et al. 2005). The main endophytic fungi work in literature involves screening for secondary metabolites of antimicrobial and antioxidant activity. Not many explored the possibility of endophytic fungi as industrially essential biotechnological reservoirs of enzymes.

    Cellulases have been widely used in agricultural, biofuel, detergent, fermentation, food, paper pulp, and textile industries (Kuhad et al. 2011). Screening of the isolates for cellulase activity was attempted with a view of endophytes penetrating the plant tissue through the lignocellulosic wall with the help of the hydrolytic enzymes, cellulases being predominant among them (Carroll & Petrini 1983). In addition, it was reported that some endophytes might behave as latent saprophytes, and when the host dies, they use these enzymes for tissue degradation to obtain nutrients (De Aldana et al. 2013). Studies also estimated that microbial pectinase accounts for 25% of global food and industrial enzymes revenues and is increasingly growing in the market (Oumer 2017). In addition, enzymes are a well-established global industry that is expected to hit USD 6.3 billion in 2021 (Oumer & Abate 2018).

    The current results revealed that 78.3% of total isolates could hydrolyze pectin in submerged fermentation, of which 77.14% were Aspergillus isolates, 76.9% Alternaria, and 86.95% Fusarium showed positive results. The present findings were in concurrence with those of Sunitha et al. (2013) who reported that 62% of their tested fungi were pectinase producers, and better than results obtained by Shubha & Srinivas (2017) who found that 30% of their tested fungi had the pectinolytic activity. However, Choi et al. (2005) have reported that pectinase production was absent in all the endophytic fungi of Brucea javanica.

    Aspergillus species was superior in pectinase activity with A. terreus being the potent strain giving rise to 163.244 IU/ml which is more than the result of pectinase production (106.7 IU/ml) produced by Aspergillus sp. Gm (KC et al. 2020) and much more the outcome of pectinase production (1.524 IU/ml) stated by (Sopalun & Iamtham 2020) from endophytic fungi isolated from Thai Orchids.

    The production of plant cell-wall digestive enzymes is now a focus of current research. Many such researches have been done into the production of cellulase and pectinase due to the huge number of application scenarios of these enzymes (Jalis et al. 2014, Edor et al. 2018, Ismail et al. 2018, Li et al. 2020, Xue et al. 2020).

  • The current research investigates the ecology of endophytic fungi in wild medicinal plants in the New Valley Governorate, Egypt and determines their ability to produce hydrolyzing enzymes. The study managed to retrieve a total of 120 fungal isolates from just eight plants, indicating their widespread distribution. The study also confirmed the ability of these fungal isolates to produce pectinase and cellulase. The potent strains of Aspergillus was the superior in enzymes production with A. terreus AUMC 14287 for CMCase and avicellase, and A. terreus AUMC 14278 for pectinase. The study further highlights the promising ability to produce extracellular enzymes by endophytic fungi, thus showing the importance of further analysis to resolve key issues in this area.

  • The authors have not declared any conflict of interests.

Figure (2)  Table (5) References (53)
  • About this article
    Cite this article
    MA Abdel-Sater, AMA Abdel-Latif, DA Abdel-Wahab, OA Al-Bedak. 2021. Endophytic mycobiota of wild medicinal plants from New Valley Governorate, Egypt and quantitative assessment of their cell wall degrading enzymes. Studies in Fungi 6(1):78−91 doi: 10.5943/sif/6/1/4
    MA Abdel-Sater, AMA Abdel-Latif, DA Abdel-Wahab, OA Al-Bedak. 2021. Endophytic mycobiota of wild medicinal plants from New Valley Governorate, Egypt and quantitative assessment of their cell wall degrading enzymes. Studies in Fungi 6(1):78−91 doi: 10.5943/sif/6/1/4
  • Catalog

    • About this article

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return