ARTICLE   Open Access    

Molecular identification and in vitro interaction of molds associated with dry rot of potato (Solanum tuberosum L.) collected in La Trinidad, Benguet, Philippines

More Information
  • Solanum tuberosum L. from the Solanaceae family is one of the most important agricultural crops grown in the Philippines next to rice. Unfortunately, harvested potatoes were plagued with various fungal diseases resulting to occurrence of dry rot affecting the production and harvest of potatoes. Dry rot in potatoes is caused by several fungal species in the genus Fusarium. Hence, this study aimed to isolate and identify fungal species associated with dry rot disease of potato. Potatoes showing diseased symptoms of dry rot were randomly selected and collected from various traders in La Trinidad, Benguet and were subjected for fungal isolation through serial dilution and plating. Identification was done through observation of cultural and morphological characteristics as well as DNA barcoding using ITS region. A total of six fungal species were subjected into BLAST which revealed the following species: Aspergillus flavus (100.00%), A. fumigatus (100.00%), A. niger (99.82%), Fusarium oxysporum (95.06%), F. solani (100.00%) and Mucor velutinosus (96.45%). To establish possible fungal species as potential biocontrol agent that would lessen the use of harmful chemicals, the identified fungal organisms were interacted with one another through in vitro interaction. Results showed that fungi exhibited antagonistic interaction against each other. Hyphal denaturation, hyphal penetration, hyphal coiling and cell lysis, were observed through microscopic observation. Meanwhile, M. velutinosus showed the ability to penetrate the host hyphae of the F. solani and F. oxysporum. Therefore, Mucor velutinosus could be a potential biological control agent against Fusarium species, which can reduce the use of harmful fungicides in controlling fungal diseases in potato that causes dry rot.
  • Huangqin tea (HQT, Fig. 1), also called Huangjin tea in Chinese, has a long history of consumption in China[1]. It is a healthy herbal tea crafted from the aerial parts of Scutellaria baicalensis Georgi, S. scordifolia Fisch, S. amoena C. H. Wrigh, and S. viscidula Bung[24]. Unlike traditional tea made from Camellia sinensis (L.) O. Kuntze, HQT lacks stimulating components and is renowned for clearing heat and dry dampness, eliminating toxins, promoting digestion, and soothing fire[1,5]. It can be enjoyed both as a hot beverage and as a cold drink, and modern research suggests that HQT exhibits several pharmacological properties, such as anti-inflammatory, chemopreventive effects of colorectal cancer[6], anti-aging[7], cardiovascular protection, hypoglycemic effect, hypolipidemic effect, anti-tumor, anti-bacterial, anti-influenza virus, and enhance human resistance[812]. The predominant compounds found in HQT are flavonoids and essential oils and its potential health benefits are likely attributed to the presence of distinctive flavonoids, including isocarthamidin-7-O-β-D-glucuronide, carthamidin-7-O-β-D-glucuronide, apigenin-7-O-β-D-glucopyranside, chrysin-7-O-β-D-glucuronide, scutellarin, baicalin, wogonoside, and chrysin[1318].

    Figure 1.  The aerial parts of S. baicalensis Georgi, dried and brewed Huangqin tea.

    HQT is made using simple ingredients and has a straightforward preparation method. Traditionally, the above-ground portions of Scutellaria species have been utilized for crafting HQT, with the differentiation between stems and leaves remaining unessential. During the hot summer season (July to August), the aerial parts (stem, leaves, flowers) of these species of Scutellaria from a single source are collected and cut into small sections, which are then directly dried for later use. Alternatively, freshly harvested branch and leaf sections may undergo a transformative process through multiple rounds of steaming and subsequent drying within a steamer. Once suitably conditioned, they find their place within sealed containers, poised for extended storage periods. The brewing ritual commences by incorporating 7−8 g of this tea into 2 L of water, with subsequent replenishments of water at intervals 2−3 times. As the brewing ritual concludes, HQT graces the senses with a resplendent golden infusion—earning it another name, 'Huangjin tea'.

    HQT boasts a range of health advantages and is frequently gathered from the upper sections of Scutellaria species plants cultivated in the vicinity, intended for tea consumption. In recent years, large planting bases have been established in several regions, such as Beijing, Inner Mongolia Wuchuan, Yakeshi, Hebei Chengde, Shanxi Province, and Shandong Province, to comprehensively develop S. baicalensis resources. HQT has gained more attention and is now produced by specialized operating companies in various grades, such as bulk tea, bagged tea, and unique tea culture[12].

    To provide up-to-date information on the chemical composition, bioactivity, and safety aspects of the Scutellaria genus from HQT, this review paper has collected related literature from various databases, such as PubMed, Web of Science, Sci Finder, Scopus, Baidu Scholar, China National Knowledge Internet (CNKI), Wanfang and Weipu Data. This paper aims to draw attention to the need for further research and application of HQT in preventing and managing certain chronic diseases.

    HQT can be derived from several Scutellaria species. S. baicalensis is the most extensively cultivated, with a significant biomass in its aerial parts. Consequently, the primary source of HQT is the aerial part of S. baicalensis, and research focused on these aerial parts is also the most extensive.

    The aerial part and the root of S. baicalensis share similarities in their primary constituents, notably the significant presence of flavonoids, which are regarded as the main active components. However, there are specific chemical differences between the above-ground and underground parts of S. baicalensis. Our preliminary experiments have revealed the disparities in chemical composition between these two parts[18]. Notably, the root of S. baicalensis finds its primary use in medicinal applications, characterized by notably higher expression levels of specific 4′-deoxyflavones compared to the aerial organs. In contrast, the above-ground parts of the plant are employed to prepare tea. This differentiation in utilizing these plant components reflects the accumulated wisdom of generations cultivated through extensive periods of tasting and experience.

    This section provided an in-depth review of the chemical composition of the plant from which HQT is derived. Additionally, a specific emphasis on reviewing the aerial parts of S. baicalensis, which serve as the primary source of HQT has been placed.

    In the 1970s, researchers began to study the chemical composition of the aerial parts of S. baicalensis. The flavonoids are the most abundant chemical components in the aerial parts (flowers, stems, and leaves), and about 54 flavonoid compounds were identified (Table 1, Fig. 2). Most of them are flavonoids, dihydro flavonoids, and glycosides. The main glycoside-forming sugars are arabinose, glucose, and glucuronide, with the most abundant glucuronide glycosides. The optimization of extraction processes can yield a remarkable total flavonoid content of up to 5% in the stems and leaves of S. baicalensis[19].

    Table 1.  Flavonoids of HQT.
    NumberNameFormulaSpeciesRef.
    12',5-Dihydroxy-3',6,7,8-tetramethoxyflavoneC19H18O8S. baicalensis[17]
    22',6-DihydroxyflavoneC15H10O4S. baicalensis[16]
    33',4',5,5',7-PentamethoxyflavoneC20H20O7S. baicalensis[16]
    44',5-Dihydroxy-3',5',6,7-tetramethoxyflavoneC19H18O8S. baicalensis[22,27]
    54',5-Dihydroxy-7-methoxyflavanoneC16H14O5S. baicalensis[16]
    65,4′-Dihydroxy-6,7,8,3′-tetramethoxyflavoneC19H18O8S. baicalensis[17]
    75,2′-Dihydroxy-6,7,8-trimethoxyflavoneC18H16O7S. baicalensis[17]
    85,2′-Dihydroxy-6,7,8,3′-tetramethoxyflavoneC19H18O8S. baicalensis[17]
    95,2′-Dihydroxy-7,8-dimethoxyflavoneC17H14O6S. baicalensis[17]
    105,2′-Dihydroxy-7,8,6′-trimethoxyflavoneC18H16O7S. baicalensis[17]
    115,6,7,3',4'-Pentahydroxyflavone-7-O-glucuronideC21H20O13S. baicalensis[13]
    125,6,7,4'-TetrahydroxydihydroflavoneC15H12O6S. baicalensis[17]
    135,6,7-Trihydroxy-4'-methoxyflavoneC16H12O6S. baicalensis[15]
    145,7-Dihydroxy-6-methoxyflavanonC16H14O5S. baicalensis[17]
    155,7,4′-Trihydroxy-6-methoxyflavanoneC16H14O6S. baicalensis[22]
    165,7,4'-TrihydroxyflavanoneC15H12O6S. baicalensis[13]
    17(2S)-5,7,8,4'-Tetrahydroxyflavanone 7-O-β-D-glucuronopyranosideC21H20O12S. baicalensis[21]
    18(2S)-5,6,7,4'-Tetrahydroxyflavanone 7-O-β-D-glucuronopyranosideC21H20O12S. baicalensis[21]
    196-Hydroxyluteolin-7-O-glucuronideC21H18O13S. baicalensis[13]
    207-MethoxychrysinC16H14O4S. baicalensis[16]
    21ApigeninC15H10O5S. baicalensis, S. amoena,
    S. scordifolia, S. viscidula
    [17,2125]
    22Apigenin-4'-glucopyransideC21H20O10S. baicalensis[17]
    23Apigenin-6-C-glucoside-8-C-arabinosideC26H28O14S. baicalensis[13]
    24Apigenin-7-O-β-D-glucopyransideC21H20O10S. baicalensis[17]
    25Apigenin-7-O-β-D-glucuronideC21H18O11S. baicalensis, S. amoena,
    S. scordifolia
    [13,23,24]
    26Apigenin-7-O-methylglucuronideC22H20O11S. baicalensis[28]
    27BaicaleinC15H10O5S. baicalensis, S. amoena,
    S. scordifolia, S. viscidula
    [2225]
    28Baicalein-7-O-D-glucopyransideC21H20O10S. baicalensis[22]
    29BaicalinC21H18O11S. baicalensis, S. amoena,
    S. scordifolia, S. viscidula
    [16,2325]
    30CarthamidinC15H12O6S. baicalensis[13,20]
    31Carthamidin-7-O-β-D-glucuronideC21H20O12S. baicalensis[22]
    32ChrysinC15H10O4S. baicalensis, S. amoena,
    S. scordifolia
    [13,2124]
    33Chrysin-7-O-β-D-glucuronideC21H20O9S. baicalensis, S. amoena[23,29]
    34DihydrobaicalinC21H20O11S. baicalensis[22,28]
    35Dihydrooroxylin AC21H20O11S. baicalensis[13]
    36GenkwaninC16H12O5S. baicalensis[16]
    37IsocarthamidinC15H10O6S. baicalensis[20,28]
    38Isocarthamidin-7-O-β-D-glucuronideC21H20O12S. baicalensis[28]
    39IsoschaftsideC26H28O14S. baicalensis[13,16]
    40IsoscutellareinC15H10O6S. baicalensis[21,30]
    41Isoscutellarein 8-O-β-D-glucuronideC21H18O12S. baicalensis[21]
    42Kaempferol 3-O-β-D-glucopyranosideC21H20O11S. baicalensis[13,28]
    43LuteolinC15H10O6S. baicalensis[22,30]
    44Norwogonin-7-O-glucuronideC21H18O12S. baicalensis, S. amoena[13,17,23]
    45Oroxylin AC16H12O5S. baicalensis, S. amoena[17,23]
    46Oroxylin A-7-O-D-glucopyransideC22H22O10S. baicalensis[22,28]
    47Oroxylin A-7-O-β-D-glucuronideC22H20O11S. baicalensis, S. amoena[13,23]
    48PinocembrinC16H12O5S. baicalensis[13]
    49Pinocembrin-7-O-glucuronideC21H20O11S. baicalensis[13,16,22]
    50SalvigeninC18H16O6S. baicalensis[21]
    51ScutellareinC15H10O6S. baicalensis[28]
    52ScutellarinC21H18O12S. baicalensis, S. amoena,
    S. scordifolia, S. viscidula
    [2325,29]
    53WogoninC16H12O5S. baicalensis, S. amoena,
    S. scordifolia, S. viscidula
    [2125]
    54WogonosideC22H20O11S. baicalensis, S. viscidula[13,25]
     | Show Table
    DownLoad: CSV
    Figure 2.  Chemical structure of flavonoids in HQT.

    In 1976, Takido et al.[20] isolated two flavanone derivatives, carthamidin and isocarthamidin, for the first time as natural products from the leaves of S. baicalensis. Later, Yukinori et al.[21] identified two new flavanones, (2S)-5,7,8,4'-tetrahydroxyflavanone 7-O-β-D-glucuronopyranoside and (2S)-5,6,7,4'-tetrahydroxyflavanone 7-O-β-D-glucuronopyranoside), in the leaves of S. baicalensis. Eight compounds of chrysin, wogonin, apigenin, salvigenin, scutellarein, isoscutellarein, apigenin 7-O-glucuronide, and isoscutellarein 8-O-glucuronide were also isolated. Wang et al.[15] used column chromatography to isolate seven flavonoids (wogonin, chrysin, 5,6,7-trihydroxy-4'-methoxyflavone, carthamindin, isocarthamidin, scutellarein, and chrysin 7-O-β-D-glucuronide) from a water extract of the leaves of S. baicalensis. Liu et al.[13] identified 21 flavonoids in the stems and leaves of S. baicalensis by HPLC-UV/MS and NMR, and found one flavonone (5,6,7,3',4'-Pentahydroxyflavone-7-O-glucuronide) was a new compound. Zhao[17] firstly isolated 5,6,7,4′-tetrahydroxyflavanone 7,5,7-dihydroxy-6-methoxyflavanone, oroxylin A, 5,4′-dihydroxy-6,7,8,3′-tetramethoxyflavone, 5,2′-dihydroxy-6,7,8,3′-tetramethoxyflavone, 5,2′-dihydroxy-7,8,6′-trimethoxyflavone, 5,2′-dihydroxy-7,8-dimethoxyflavone, 5,2′-dihydroxy-6,7,8-trimethoxyflavone, apigenin 4'-β-D-glucopyranoside, and apigenin-7-β-D-glucopyranoside from the aerial parts of S. baicalensis. Ma[22] firstly isolated 5,7,4'-trihydroxy-6-methoxyflavone, 5,4'-dihydroxy-6,7,3',5'-tetramethoxyflavone, from stems and leaves of S. baicalensis. Wang et al.[16] isolated 5,4'-dihydroxy-7-methoxyflavanone, genkwanin, 7-methoxychrysin, 3',4',5,5',7-pentamethoxyflavone from 60% ethanol extracts for stems and leaves of S. baicalensis for the first time. Also, the compounds of carthamidin-7-O-β-D-glucuronide, oroxylin A-7-O-β-D--glucuronide, and chrysin were isolated from this plant for the first time.

    The concentration of these chemical components in HQT varies depending on the plant part utilized. Employing the HPLC-DAD method, Shen et al.[18] established that the aerial parts (stems, leaves, and flowers) of S. baicalensis are rich in flavonoids, resembling the roots in composition but exhibiting significant disparities in content. The contents of isocarthamidin-7-O-β-D-glucuronide (106.66 ± 22.68 mg/g), carthamidin-7-O-β-D-glucuronide (19.82 ± 11.17 mg/g), and isoscutellarein-8-O-β-D-glucuronide (3.10 ±1.73 mg/g) were the highest in leaves. The content of apigenin-7-O-β-D-glucopyranoside (18.1 ± 4.85 mg/g) and chrysin-7-O-β-D-glucuronide (9.82 ± 5.51 mg/g) were the highest in flowers. HQT has a high content proportion of flavone glycosides, which is closely related to the activity of HQT. The concentrations of the nine main flavonoids in HQT infusions were measured using HPLC. The content of isocarthamidin-7-O-β-D-glucuronide (52.19 ± 29.81 mg/g) was the highest; carthamidin-7-O-β-D-glucuronide (31.48 ± 6.82 mg/g), chrysin-7-O-β-D-glucuronide (10.65 ± 0.40 mg/g) and apigenin-7-O-β-D-glucopyranside (5.39 ± 0.92 mg/g) were found at moderate levels in HQT samples. As for flavone aglycones, scutellarin (12.77 ± 1.14 mg/g), baicalin (1.88 ± 0.48 mg/g), isoscutellarein-8-O-β-D-glucuronide (2.84 ± 0.60 mg/g), wogonoside (0.23 ± 0.02 mg/g) and chrysin (0.03 ± 0.01 mg/g) has lower content in HQT[6].

    Although there are few studies on the chemical constituents of the aerial parts of S. amoena, S. scordifolia, and S. viscidula it has been shown that the compounds of the aerial parts are similar to S. baicalensis. The aerial parts of S. amoena contain the compounds of baicalein, baicalin, oroxylin A, oroxylin A-7-O-β-D-glucuronide, wogonin, chrysin, chrysin-7-O-β-D-glucuronide, norwogonin, 5,7-dihydroxy-6,8-dimethoxyflavone, scutellarin[23]. Zhang et al.[24] identified compounds of chrysin, wogonin, baicalein, apigenin, apigenin-7-O-β-D-glucoside, baicalin, and scutellarin in whole plants of S. scordifolia. The stems and leaves of S. viscidula all contain compounds of wogonoside, apigenin, baicalein, wogonin, baicalin, and scutellarin. The contents of baicalein, wogonoside, wogonin, and apigenin in the stem of S. viscidula were higher than those in the stem of S. baicalensis. In the leaves of the two species, the content of scutellarin was higher, while the content of other compounds was lower[25]. The content of scutellarin in S. viscidula was stem (2.30%) > leaf (1.78%) > flowers (0.38%)[26].

    The aerial parts of S. baicalensis are rich in essential oils, and the taste of HQT is closely related to this. The flowers of S. baicalensis are thought to have a Concord grape aroma, while HQT has a bitter flavor with distinctive herbal notes. Extensive analysis has identified 145 components in the essential oil obtained from the aerial parts of S. baicalensis. These components span various chemical classes, such as alkanes, carboxylic acids, fatty acids, monoterpenes/oxygenated monoterpenes, sesquiterpenes triterpenoids and Vitamins (Supplemental Table S1), which have demonstrated their efficacy in combatting bacteria, reducing inflammation and inhibiting tumor growth[2731]. Among these, major constituents include germacrene D (5.4%−39.3%), β-caryophyllene (29.0%), caryophyllene (18.9%), eugenol (18.4%), caryophyllene (15.2%), caryophyllene oxide (13.9%), (E)-β-caryophyllene (11.6%), 5-en-3-stigmasterol (11.3%), carvacrol (9.3%), thymol (7.5%), vitamin E (7.4%), neophytadiene (7.3%), γ-elemene (6.2%), 1-octen-3-ol (6.1%), allyl alcohol (5.5%), bicyclogermacrene (4.8%), myristicin (4.7%), acetophenone (4.6%), α-amyrin (4.6%), β-amyrin (4.4%), germacrene d-4-ol (4.3%), spathulenol (4.2%), β-pinene (4.1%), α-humulen (4.0%), 1-vinyl-1-methyl-2-(1-methylvinyl)-4-(1-methylethylidene)-cyclohexane (4.0%) are found in the aerial parts of S. baicalensis from different places[2731].

    Takeoka et al.[27] identified 64 components in volatile components of S. baicalensis flowers by solid-phase microextraction and analyzed them by GC and GC-MS. These flowers were collected at San Francisco State University (USA). Among the flower volatiles, the content of β-caryophyllene, germacrene D, δ-cadinene, γ-muurolene, and γ-cadinene were more than 3%. The essential oil obtained from the stem of S. baicalensis is mainly composed of diphenylamine, 2,2-methylenebis (6-tert-butyl-4-methylphenol), bornyl acetate, β-caryophyllene, germacrene D and 1-octen-3-ol.[32]. Gong et al.[28] analyzed and identified the specific chemical constituents of the aerial parts of S. baicalensis by using GC-MS technology and identified 37 compounds in total, such as allyl alcohol, acetophenone, caryophyllene, α-humulene, germacrene D, and γ-elemene. The plant material was collected in the Qinling Mountains in China. Lu et al.[29] found a big difference in essential oil components between the aerial and root of S. baicalensis from Kunming Botanical Garden, Yunnan Province (China). The aerial part of S. baicalensis mainly contained enols and sterols such as neophytadiene and vitamin E. However, it has the same compounds as the roots, such as nerolidol, hexadecanoic acid, 1,2-benzenedicarboxylic acid, squalene, stigmast-4-en-3-one, and partial alkanes. Recently, Wang et al.[31] found the essential oil level of the aerial parts of S. baicalensis was 0.09% (v/w, based on fresh weight) while its density was 0.93 g/mL, and obtained 31 components accounting for 97.64% of the crude essential oil, including sesquiterpenoid, monoterpenoids, phenylpropanoids, and others. It is also reported that the major components of the essential oil from the aerial parts of S. baicalensis were myristicin, eugenol, caryophyllene, caryophyllene oxide, germacrene D, spathulenol, and β-pinene, with eugenol as the most abundant. The sample of the aerial parts were harvested from Tangshan City (China). The composition of S. baicalensis essential oils varies according to the plant part used, geographical location, and growing conditions.

    Zgórka & Hajnos[33] identified the phenolic acid compounds of aerial parts of S. baicalensis by solid-phase extraction and high-speed countercurrent chromatography: p-coumaric acid, ferulic acid, p-hydroxybenzoic acid, and caffeic acid. Chirikova & Olennikov[34] found that the aerial part of S. baicalensis contains 11 kinds of saturated fatty acids and nine kinds of unsaturated fatty acids, among which the palmitic acid content is the highest. Chlorogenic acid, fernlic acid, protocatechuic acid, vanillic acid, rosmarinic acid, caffeic acid, p-hydroxybenzoic acid, and p-coumaric acid were also detected.

    Zhao [17] isolated four sterol compounds: β-sitosterol-3-O-β-D-glucoside, α-apinasterol, β-sitosterol, and four ester compounds: methoxyphaeophorbide, p-hydroxybenethyl ethanol hexadecanoic methyl ester, ethoxyphaeophorbide, and n-octadecanol, lutein from the aerial parts of S. baicalensis.

    It is reported that flavonoids and diterpenes are the two main groups of active constituents in the genus Scutellaria. However, only one diterpene (scutebaicalin) was identified in the stems and leaves of S. baicalensis[35].

    By atomic absorption spectrophotometry, Yuan et al.[36] determined the contents of 11 metal elements in different parts of S. baicalensis. It was found that the leaves and stems of S. baicalensis were rich in Mg, K, Cr, Ni, Co, Fe, Mn, and Pb. Meanwhile, Yan et al.[37] developed an inductively coupled plasma mass spectrometry method and determined 23 kinds of inorganic elements in the stems and leaves of S. baicalensis from eight regions. Although there were no differences in the types of inorganic elements in the stems and leaves of S. baicalensis from the different areas, the content of these elements varied significantly. Among these elements, Fe, Zn, Cu, Mn, Cr, Co, Ni, Sr, B, and Ni were essential human body elements. The content of Al (516.83 μg/g) and Fe (700.62 μg/g) was the highest, while the content of B (31.54 μg/g), Ti (23.10 μg/g), Mn (65.64 μg/g), Sr (62.27 μg/g), and Ba (89.68 μg/g) was relatively high.

    Olennikov et al.[38] studied the water-soluble polysaccharides from the aerial parts of S. baicalensis from Russia and found that the polysaccharides from S. baicalensis gradually accumulated before flowering and progressively decreased after flowering.

    Yan et al.[39] found that the stems and leaves of S. baicalensis were rich in amino acids, and there was no difference in the kinds of amino acids among different producing areas, but there was a significant difference in the contents of amino acids. The content of proline, threonine, glutamic acid, lysine, glutamine, and arginine was higher, and the content of methionine, hydroxyproline, and citrulline was low.

    Several studies have focused on the functional properties of HQT, with increasing attention given to the aerial parts of S. baicalensis as the main raw material for HQT production. S. baicalensis stems and leaves flavonoids (SSF) are considered the functional components of HQT. Modern pharmacology has shown that the flavonoids extracted from the stem and leaf of S. baicalensis have been found to possess anti-inflammatory, anti-bacterial, antiviral, antipyretic and analgesic, anti-tumor, hepatoprotective, antioxidant, hypoglycemic, hypolipidemic, detoxification, myocardial ischemia protection, brain injury protection, and immunomodulatory effects. However, few studies have been conducted on the individual flavonoid compounds in the total flavonoid extract from S. baicalensis. Therefore, this paper aims to summarize and supplement the current research on the functional properties of HQT and its primary raw material (S. baicalensis) extract.

    Injury and infection could lead to inflammation, which plays a key role in the accelerated pathogenesis of immune-mediated disease[40]. Tong et al.[41], Zhou et al.[42], and Zhao et al.[43] found that S. baicalensis stem-leaf total flavonoid (SSTF) could inhibit acute exudative inflammation caused by xylene, glacial acetic acid, and egg white and also have a significant inhibitory effect on chronic inflammatory of granulation tissue hyperplasia. Wang et al.[44] observed the effect of SSTF on the aerocyst synovitis of the rat model and found that it could reduce capillary permeability, reduce the aggregation of neutrophils and basophils in tissues, reduce histamine, bradykinin, and other substances that increase vascular permeability, which is conducive to the recovery of vascular permeability in inflammation. Studies have shown that the SSTF significantly inhibits specific and non-specific inflammatory responses and can regulate the body's cellular and humoral immune functions. The mechanism of action is closely related to the effective reduction of capillary permeability, inhibition of PGE2 and NO synthesis in vivo, reduction of TNF-α expression, and reduction of inflammatory exudation[45,46]. SSTF (200 mg/kg) could balance the CD+4 Tlymphocyte subsets Th1/Th2 cells and the related cytokines IL-10 and IFN-γ in the rheumatoid arthritis model[47]. SSTF (17.5, 35 and 70 mg/kg for 38 d) could significantly improve the impairment of relearning ability and retention ability on memory impairment and nerve inflammation in chronic cerebral ischemia rats, which might be due to the inhibition of the proliferation of astrocyte and balanced the expression of the inflammatory factors in the brain[48]. Besides, the extract of S. baicalensis stem-leaf shows anti-inflammation effects both in vitro and in vivo. In cultured macrophage cells (RAW 264.7), the extract of S. baicalensis stem-leaf showed a strong anti-inflammation effect, which inhibited the expression of IL-1β. Similarly, it suppressed the LPS-induced transcriptional activity mediated by NF-κB in fish aquaculture[49]. S. baicalensis stems and leaves (3, 6, and 12g /kg, gavage for 7 d, once per day) have anti-inflammatory effects on 2% carrageenan-induced acute pleurisy in rats, and the mechanism of action may be related to the reduction of the production of inflammatory factors and the down-regulation of TRPV1 signaling protein[50]. The combination of S. baicalensis stems- Polygonum cuspidatum (3.5, 7, and 14 g/kg, gavage for 7 d) has a protective effect on lipopolysaccharide-induced acute lung injury rats, and its mechanism may be related to down-regulating the expression of TRPV1 and inhibiting the levels of TNF-α and IL-1β in inflammatory cells[50]. It is also reported that S. baicalensis stem-leaf combined with Morus alba (4, 8, and 16 g/kg/d, gavage, 10 d) has a protective effect on rats with acute pneumonia induced by lipopolysaccharide, and the mechanism might be related to the reduction of inflammatory factors and the down-regulation of TRPV1 signaling pathway[51]. Besides, the network pharmacology showed that S. baicalensis stem-leaf could prevent and control COVID-19 by intervening in 30 targets and 127 pathways, potentially preventing and treating inflammation caused by COVID-19[52].

    Based on the studies, S. baicalensis stem-leaf extract shows promising anti-inflammatory properties. These effects are likely mediated through a combination of factors, including the modulation of immune responses, reduction of inflammatory mediators, and potential interactions with signaling pathways like TRPV1. However, it's important to note that while these studies provide valuable insights, further research, including clinical trials, is needed to establish the full extent of its benefits and its potential for therapeutic applications in humans.

    Scutellaria baicalensis stem and leaf aqueous extract exhibit different degrees of inhibition of 36 strains from 13 kinds of bacteria, such as Staphylococcus aureus, Staphylococcus, Streptococcus pneumoniae, alpha-hemolytic streptococcus, beta-hemolytic streptococcus and Escherichia coli. This shows that anti-bacterial activity against Staphylococcus aureus is strong (MIC50 0.94 g/L, MBC 0.94 g/L). In vivo (217 mg/kg), it protects against the death of mice infected by Staphylococcus aureus and shows a certain dose-dependence[53]. Zhang et al.[54] found that the stem and leaves of S. baicalensis against Staphylococcus aureus and Shigella dysenteriae with MIC values of 1 and 4 mg/mL, respectively. Besides, it is reported that the water extract of the aerial part of S. baicalensis could inhibit the growth of several common pathogenic bacteria in aquacultures, such as Aeromonas hydrophila, Edwardsiella tarda, Vibrio alginolyticus and V. harveyi[49].

    Zhao et al.[55] found that the active part of the stem and leaf of S. baicalensis could inhibit the cytopathic effect caused by 10 kinds of viruses such as Coxsackie B virus, influenza virus, parainfluenza virus, adenovirus, respiratory syncytial virus, and herpes simplex virus. It is suggested that the active parts of the stem and leaf of S. baicalensis can be used for the prevention and treatment of influenza virus, parainfluenza virus, coxsackievirus, and other related infectious diseases.

    These findings indicate that S. baicalensis stem and leaf extract possess anti-bacterial and antiviral properties, making it a potentially valuable natural resource for combating infections caused by various pathogens. However, while these results are promising, further research, including clinical trials, would be necessary to fully establish the effectiveness and safety of using S. baicalensis extract for preventing or treating bacterial and viral infections, including COVID-19.

    In a series of studies, Tong et al.[41] demonstrated that SSTF at a dosage of 20 mg/kg significantly reduced body temperature in rats with fever induced by subcutaneous injection of a 10% dry yeast suspension. Zhang et al.[56] conducted research on the antipyretic effect of scutellarin, an extract from S. baicalensis stems and leaves, in febrile rabbits and observed an antipyretic substantial impact induced by pyrogen. Yang et al.[57] conducted several animal experiments, where they discovered that intraperitoneal injection of effective doses of SSTF (42.2 and 84.4 mg/kg) exhibited a specific inhibitory effect on infectious fever in experimental animals. Moreover, intraperitoneal injection of appropriate SSTF doses (30.1, 60.3, and 120.6 mg/kg), as found in Yang et al.'s experiments[58], effectively inhibited the pain response in experimental animals. Furthermore, Zhao et al.[59] noted that SSTF exhibited a certain inhibitory effect on the pain response in experimental animals subjected to chemical and thermal stimulation.

    These findings suggest that S. baicalensis stem and leaf extract may have antipyretic and analgesic effects, particularly its total flavonoid component. These effects could be beneficial for managing fever and providing pain relief. However, as with any natural remedy, further research, including controlled clinical trials in humans, is necessary to fully understand the effectiveness, safety, and optimal dosing of S. baicalensis extract for these purposes.

    Amyloid protein (Aβ) has been widely recognized as the initiator of Alzheimer's disease (AD)[60]. The SSTF can improve cognitive function and delay the process of dementia. SSTF has been found to exert neuroprotective effects in AD animal models through various mechanisms. Ye et al.[61] demonstrated that oral administration of SSTF (50 mg/kg) could effectively improve cognitive function and reduce neuronal injury in Aβ25-35-3s -induced memory deficit rats. The underlying mechanisms may involve inhibiting oxidative stress and decreasing gliosis[62,63]. Furthermore, SSTF was shown to reduce Aβ-induced neuronal apoptosis by regulating apoptosis-related proteins Bax and Bcl-2[64]. Subsequent studies further validated the neuroprotective effects of SSTF. Cheng et al.[65] found that SSTF treatment inhibited neuronal apoptosis and modulated mitochondrial apoptosis pathway in composited Aβ rats. Ding & Shang[66] found that the SSF improves neuroprotection and memory impairment in rats due to its inhibition of hyperphosphorylation of multilocus Tau protein in rat brains. SSTF has also been found to exert neurogenesis-promoting effects by regulating BDNF-ERK-CREB signaling[12] and activating the PI3K-AKT-CREB pathway[67].

    In further studies, Ding et al.[11] proposed that the effect of SSF on promoting neurogenesis and improving memory impairment may be related to the regulation of abnormal expression of Grb2, SOS1, Ras, ERK, and BDNF molecules in the BDNF-ERK-CREB signaling pathway. Zhang et al.[68] found that SSF (25, 50, and 100 mg/kg) could significantly modulate okadaic-induced neuronal damage in rats, which provides a basis for evaluating SSF as a means to reduce tau hyperphosphorylation and Aβ expression in Alzheimer's disease. Cao et al.[69] found that SSTF (100 mg/kg, 60 d) may alleviate tau hyperphosphorylation-induced neurotoxicity by coordinating the activity of kinases and phosphatase after a stroke in a vascular dementia rat model. Gao et al.[70] demonstrated that the stems and leaves of S. baicalensis (SSF, 25, 50, and 100 mg/kg/d, 43 d) could inhibit the hyperphosphorylation of tau in rats' cerebral cortex and hippocampus induced by microinjection of okadaic acid, which may be related to the activities of protein kinase CDK5, PKA and GSK3β. Furthermore, Liu et al.[67] demonstrated that SSF (35, 70, and 140 mg/kg/d, 43 d) improved composited Aβ-induced memory impairment and neurogenesis disorder in rats through activated the PI3K-AKT-CREB signaling pathway and up-regulated the mRNA and protein expression of TRKB, PI3K, AKT, CREB and IGF2. More recently, a new study demonstrated that SSF (35, 70, and 140 mg/kg) alleviated myelin sheath degeneration in composited Aβ rats, potentially modulating sphingomyelin metabolism[71]. Collectively, these findings suggest that SSTF holds therapeutic potential for AD by targeting multiple Alzheimer's pathogenesis-related processes.

    Li et al.[72] confirmed that SSTF (5 mg/kg) could improve the behaviors and the numbers of dopaminergic neurons in the substantia nigra in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson's disease in mice, and these beneficial activities appear to be associated with the reduction of the level of serum malondialdehyde.

    These studies suggest that S. baicalensis stem and leaf extract have the potential to exert neuroprotective effects in various neurodegenerative conditions, including Alzheimer's disease and Parkinson's disease. These effects could be attributed to its ability to modulate oxidative stress, apoptosis, signaling pathways, and protein hyperphosphorylation. However, as with any potential therapeutic agent, further research is needed to establish the full extent of its benefits, optimal dosages, and mechanisms of action, as well as its potential applications in human patients.

    In recent years, ischemic cerebrovascular disease has seriously threatened human health. Cerebral ischemia is one of the leading causes of death. It can occur in focal or global ischemia, with most cases associated with ischemic stroke[73]. Neuronal protection against oxidative damage has been proposed as a potential therapeutic strategy to avoid damage during ischemic stroke[74]. It is reported that SSTF can reduce neuronal apoptosis and free radical damage caused by heart and brain ischemia. Zhao et al.[75] have found that the pretreatment of SSTF (100 mg/kg/d) can protect the ischemia-reperfusion myocardium by enhancing the activity of the anti-oxidative enzyme, inhibiting lipid peroxidation and attenuating the oxygen-free radicals-mediated damage to the myocardium in rats. In further studies, Zhao et al.[76] proposed that SSTF (50, 100, or 200 mg/kg/d, 7 d) pretreatment could alleviate the neuronal damage incurred by ischemia-reperfusion, demonstrating a neuroprotective effect in focal ischemia-reperfusion rat model, which may involve the prohibition of the apoptosis of the neurons. Yu et al.[77] confirmed that SSTF (17.5, 35, and 70 mg/kg/d, 7 d) could attenuate cardiomyocyte apoptosis during ischemia reperfusion injury by down-regulating the protein expression of the JAK2 gene. Qin et al.[78] found that SSF (17.5, 35, and 70 mg/kg/d, 38 d) can decrease the expression of the NMDAR in hippocampus, and increase the expression of VEGF in the cerebral cortex of chronic cerebral ischemia rats.

    Focal cerebral ischemia-reperfusion can result in neuronal loss but strongly promotes activation and proliferation of hippocampal glial cells. Losing hippocampal neurons is considered one of the basic pathological mechanisms of cognitive impairment[79]. Zhao et al.[79] found that the pretreatment with SSTF (100 and 200 mg/kg) could improve neurological function after focal cerebral ischemia-reperfusion injury, with preventive and protective effects. Shang et al. found flavonoids from S. baicalensis (35−140 mg/kg) could attenuate neuron injury and improve learning and memory behavior in rats with cerebral ischemia/reperfusion[80]. In further studies, Kong et al.[81] found that the mechanisms of the protective effects on the brain against cerebral ischemia/reperfusion injury of SSTF may involve decreasing the content of brain water, increasing microvascular recanalization, reducing the apoptosis of hippocampal neurons, and attenuating free radical damage. Bai et al.[82] proposed that SSTF (100 mg/kg/d, 7 d) could protect the neurological function in rats following I/R injury by alleviating the damage to the ultrastructure of cerebral cortex neurons and synapse. Yan et al.[83] found that SSTF (100 mg/kg/d, 7 d) pretreatment can exert preventive, protective effects on cerebral tissue by relieving brain edema, decreasing neural damage, promoting microvascular repatency, and increasing enzyme activity. It has been reported that the SSTF may protect neurons and their synaptic structures in multiple ways, but whether this mechanism enhances the resistance of neurons to damage or increases the repair function remains to be further explored.

    Essential hypertension is a common chronic cardiovascular disease, which can lead to multiple target organ damage, such as heart, brain, and blood vessels. It is a risk factor for coronary heart disease, heart failure, and other cardiovascular diseases. It is reported that SSTF (17.5, 35.0, and 70 0 mg/kg, 8 weeks) can inhibit myocardial remodeling in primary hypertensive rats, and the medium dose exerts the best inhibitory effect, and the mechanism may be related to inflammatory response induced by inhibiting the NF-κB signaling pathway[84].

    S. baicalensis stem and leaf extract have the potential to provide neuroprotective effects in conditions related to ischemic cerebrovascular disease and hypertension. Its ability to modulate oxidative stress, inflammation, and apoptotic pathways appears to contribute to its beneficial effects. However, further research is needed to fully understand the mechanisms and optimal usage of SSTF for these therapeutic purposes.

    Aging is associated with the deterioration of physiological function and the decline of cognitive ability[85]. It is reported that the alcohol extracts from roots, stems, leaves, and flowers of S. baicalensis (400 mg/kg, 7 weeks) could regulate the content of differential metabolites in urine samples of D-gal-induced aging-model rats to different degrees and play a certain role in improving the metabolic disorders of aging rats[7]. A further study investigated the anti-aging effects and potential mechanisms of S. baicalensis leaves and flower extract. S. baicalensis leaves (400 and 800 mg/kg, 7 weeks) have an anti-aging effect, which can improve the acquired alopecia, slow response, and other characteristics of aging rats, increase the spontaneous activity of aging rats, and reduce the damage of lipid peroxidation and glycosylation induced by D-galactose[86]. The S. baicalensis flowers extract (400 and 800 mg/kg, 7 weeks) could effectively reverse the cognitive decline and oxidative stress injury and alleviate liver pathological abnormalities in the D -galactose-induced aging rats, which are involved in the glutamine-glutamate metabolic pathway[85].

    S. baicalensis extracts, particularly those from leaves and flowers, may have anti-aging properties by regulating metabolic disorders, improving cognitive function, reducing oxidative stress, and alleviating aging-related physiological abnormalities. However, further research is necessary to fully understand the mechanisms underlying these effects and to determine the potential of these extracts for human applications in addressing age-related issues.

    The SSTF (200 mg/kg d, 35 d) can reduce the joint damage of collagen-induced arthritis mice and balance the CD+4 T lymphocyte subsets Th17 and Treg cells[87]. Besides, on the multiple sclerosis model, the SSTF (100, 200 mg/kg d, 16 d) displayed a protective effect on experimental autoimmune encephalomyelitis rats through a balance of the CD+4 Tlymphocyte subsets Th17 and Treg cells[88]. Zhang et al.[89] have found that SSTF attenuated EAE disease severity, accompanied by enhanced Treg frequency and level of Treg-associated cytokines (IL-10 and TGF-β), as well as downregulated Th17 frequency and expression of Th17-related cytokines (IL-17 and IL-23).

    It is reported that the essential oils from the aerial parts of S. baicalensis showed toxicity against booklice (Liposcelis bostrychophila) with an LC50 of 141.37 μg/cm2. The components of myristate, caryophyllene, eugenol, and caryophyllene oxide displayed dramatic toxicity against the L. bostrychophila, with LC50 values of 290.34, 104.32, 85.75, and 21.13 μg/cm2, respectively[31].

    Guo & Xu[90] have found that SSTF could inhibit the proliferation of Hela cells obviously (p< 0.01). Tang et al.[10] have found that SSTF could play an anti-colon cancer role by up-regulated the expressions of Cleaved Caspase-3 and the ratio of Bax/Bcl-2 (p < 0.05 and 0.01) and significantly down-regulated the expressions of MMP-2 and MMP-9 in HCT116 cells (p < 0.05 and 0.01). Recently, Shen et al.[6] studied the chemopreventive effects of HQT against AOM-induced preneoplastic colonic aberrant crypt foci in rats and found HQT inhibits AOM-induced aberrant crypt foci formation by modulating the gut microbiota composition, inhibiting inflammation and improving metabolomic disorders.

    Cardiovascular disease is one of the most important threats to human health. Hyperlipidemia is a major risk factor for atherosclerosis, which can cause various cardiovascular and cerebrovascular diseases. Different doses of SSTF (50, 100, and 200 mg/kg) can effectively reduce the body weight increase of rats with hypertriglyceridemia, reduce the serum levels of triglyceride(TG), total cholesterol(TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C), which indicate that SSTF has the effect of regulating blood lipid[91].

    Oxidative stress is important in developing tissue damage in several human diseases[92]. The antioxidant capacities of separated organs (flower, leaf, stem, and root) of S. baicalensis were conducted by DPPH, ABTS+, and RP methods, respectively. The results showed that the antioxidant activity of the root (66.9 ± 0.3, 121.6 ± 0.5, and 80.2 ± 0.4 μg/mL) was the highest, followed by the leaf (68.4 ± 1.3, 128.2 ± 2.1, and 135.8 ± 2.0 μg/mL), stem (127.8 ± 3.1, 199.2 ± 1.7, and 208.2 ± 8.3 μg/mL) and flower (129.8 ± 6.3, 285.7 ± 4.7, and 380.3 ± 14.2 μg/mL)[93]. The antioxidant activities of the extracts of the aerial part of S. baicalensis and separated organs were conducted by DPPH assay and reducing power method with the antioxidant ability 7.73–8.83 mg TE/g DW and 51.48–306.09 mg TE/g DW, respectively. The content of total polyphenols and total flavonoids were significantly positively correlated with the reducing power[94]. Liu et al.[95] found that the flavonoids extracted from the stems and leaves of S. baicalensis (SSF, 18.98, 37.36, and 75.92 μg/mL) could protect rat cortical neurons against H2O2-induced oxidative injury in a dose-dependent manner. Cao et al. found that SSTF could alleviate the damage of human umbilical vascular endothelial cells injured by H2O2 and reduce their apoptosis, which may be related to the increasing level of Bcl-2[96].

    Preventive treatment with SSTF (50, 100, and 200 mg/kg) could significantly inhibit the blood glucose increase induced by alloxan in mice, and SSTF treatment could reduce the blood glucose level in diabetic mice. Both the prevention group and the treatment group could increase the activity of serum superoxide dismutase and decrease the content of malondialdehyde[97]. Liu et al.[98] found that SSTF (75 and 150 mg/kg) could significantly reduce blood glucose and blood lipid and improve insulin resistance in type 2 diabetic rats with hyperlipidemia.

    Yang et al.[99] found that SSTF (35 mg/kg/d, 8 weeks) could resist hepatic fibrosis by inhibiting the expression of α-smooth muscle actin in Hepatic Stellate Cells. In vivo, it is reported that SSTF (50, 100, and 200 mg/kg) could significantly reduce alanine transaminase activities in serum, increase the expression of superoxide dismutase and reduce the content of malondialdehyde in acute hepatic injury mice induced by carbon tetrachloride and ethanol[100].

    These findings suggest that S. baicalensis stem and leaf extract hold promise in promoting various aspects of health, including immune modulation, antioxidant activity, anti-tumor effects, cardiovascular health, oxidative stress protection, and more. However, further research, including clinical studies, is necessary to better understand the full therapeutic potential and safety of these effects in human applications.

    Flavonoids are considered the main active components in HQT. As the active substance basis of HQT, the safety of SSTF has also been investigated. After 90 d of oral administration of SSTF (0.5, 1, and 2 g/kg) to rats, no abnormal changes were observed in all indexes, and no delayed toxic reactions or obvious toxic reactions were observed, indicating that the toxicity of SSTF is low[101]. The LD50 value of SSTF was 14.87 g/kg is equivalent to 68.5 times the maximum dose in the pharmacodynamic test of mice, and the experiment confirms the safety of oral administration of the SSTF. Intraperitoneal injection of SST showed certain toxicity in mice, with an LD50 value of 732.11 mg/kg[102]. Liu et al.[103] conducted a systematic safety assessment experiment on the aqueous extract of S. baicalensis stem and leaves based on the China National Standard 'Guidelines for the Safety Evaluation of Food Toxicology (GB15193-2014)'. The results indicated that S. baicalensis stem and leaves are non-toxic, non-teratogenic, and non-mutagenic. Acute toxicity tests in mice revealed a Maximum Tolerated Dose of 15.0 g/kg. A 90-d feeding trial showed no changes in toxicological damage in animals, even at a high dosage of 8.333 g/kg (equivalent to 100 times the recommended human daily intake), suggesting the safety and non-toxicity of consuming S. baicalensis stem and leaves. In addition, HQT has long been used in folklore, and no toxicity has been reported.

    These findings collectively indicate that HQT is generally safe for consumption. However, as with any herbal product, it's important to follow recommended dosages and consult healthcare professionals, especially for individuals with pre-existing health conditions or medications.

    In recent years, HQT, a non-Camellia tea with a long history in China, has attracted attention due to its diverse pharmacological activities. Among various Scutellaria species, S. baicalensis is the most extensively studied and cultivated source for HQT. The aerial parts, including flowers, stems, and leaves, serve as the principal source of HQT preparation. HQT are rich in flavonoids and volatile components with various beneficial effects. To date, about 295 compounds have been identified from HQT, including approximately 54 flavonoid compounds and 145 volatile components identified online. The current research on the activity of HQT primarily focuses on flavonoid compounds, with limited studies on the larger quantity of volatile oil compounds.

    Additionally, the processing and brewing techniques used to prepare HQT may influence the bioactivity of its flavonoid content, although few studies have investigated this. More research is necessary to optimize the processing and brewing techniques to maximize the health benefits of HQT. Comparative studies reveal that the aerial parts of S. baicalensis, while sharing similarities with the roots, contain varying flavonoid compositions. Limited research on S. scordifolia, S. amoena, and S. viscidula, indicates the presence of comparable flavonoid compounds in their aerial parts. Although individual flavonoids like baicalin, wogonin, and scutellarin have demonstrated various therapeutic properties, it is essential to consider the synergistic effects of these compounds when consumed together in the form of tea. These findings contribute to laying the groundwork for quality assessment of HQT and offer insights into potential health benefits.

    HQT is mainly derived from the aerial parts of S. baicalensis. Recent studies have increasingly recognized the pharmacological value of the aerial parts of S. baicalensis. Preliminary pharmacological studies have shown that the aerial parts of S. baicalensis may possess beneficial activities in antioxidant, anti-tumor, antiviral, anti-bacterial, protection of ischemia-reperfusion injured neural function, neuroprotective effects against brain injury, and blood lipid regulation. These findings suggest that the value of using HQT may be attributed to these pharmacological activities. Although HQT is generally safe for consumption, further investigation is required to understand its safety profile, particularly in special populations such as pregnant or lactating women, children, and individuals with pre-existing medical conditions. Additionally, potential interactions between the flavonoids in HQT and conventional medications must be examined to ensure their safe and effective use with pharmaceutical treatments. In addition, although the safe dose of HQT on rodents has been studied, the safe dose for humans has yet to be determined.

    In conclusion, these initial research results support the potential health benefits of HQT and encourage more in-depth studies on its raw materials. Further studies are necessary to elucidate the synergistic effects of the flavonoids in HQT, optimize the processing and brewing techniques for maximum bioactivity, and investigate the safety profile and potential interactions with conventional medications. A comprehensive understanding of HQT will contribute to developing evidence-based recommendations for promoting health and well-being.

    The authors confirm contribution to the paper as follows: Conceptualization and writing: Quan Y, Li Z, Meng X, Li P, Shen J; Figure and table modification: Quan Y, Li Z, Meng X, Li P; review and editing: Wang Y, He C, Shen J. All authors reviewed the results and approved the final version of the manuscript.

    The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

    This research was funded by the Shandong Provincial Natural Science Foundation, China (ZR2022QH147, ZR2022QH165) and the Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine (No. ZYYCXTD-D-202005).

  • The authors declare that they have no conflict of interest.

  • Adebola MO, Amadi JOE. 2010 -Screening three Aspergillus species for antagonistic activities against the cocoa black pod organisms (Phytophthora palmivora). Agriculture and Biology Journal of North 1, 362-365.

    Google Scholar

    Alvez MH, Takaki GM, Porto AL, Milanez AI. 2002 - Screening of Mucor spp. for the production of amylase, lipase, polygalacturonase and protease. Brazillian Journal of Microbiology 33.

    Google Scholar

    Arya AO, Parello AE. 2010 - Management of fungal plant pathogens. Center for Agriculture and Bioscience International Publishing, 403.

    Google Scholar

    Barnette HL. 1963 -The nature of mycoparasitism by fungi. Annual Review Microbiology 17, 1- 14. doi: 10.1146/annurev.mi.17.100163.000245

    CrossRef    Google Scholar

    Bellemain E, Carlsen T, Brochmann C, Coissac E, Taberlet P, Kauserud H. 2010 - ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases. BMC Microbiology 10, 189. doi: 10.1186/1471-2180-10-189

    CrossRef    Google Scholar

    Brimmer TA, Boland GJ. 2003 -A review of the non-target effects of fungi used to biologically control plant diseases. Agriculture, Ecosytem and Environment 100, 3-16. doi: 10.1016/S0167-8809(03)00200-7

    CrossRef    Google Scholar

    Camire ME, Kubow S, Donnely D. 2009 -Potatoes and human health. Critical Reviews in Food Science and Nutrition 49, 823-40. doi: 10.1080/10408390903041996

    CrossRef    Google Scholar

    Chamoun R, Jabaji S. 2011 -Expression of genes of Rhizoctonia solani and the biocontrol Stachybotrys elegans during mycoparasitism of hyphae and sclerotia. Mycologia Journal 103, 483-493. doi: 10.3852/10-235

    CrossRef    Google Scholar

    Chet I, Harman GE, Baker R. 1981 - Trichoderma hamatum: its hyphal interactions with Rhizoctonia solani and Pythium spp. Microbial Ecology 7, 29-38. doi: 10.1007/BF02010476

    CrossRef    Google Scholar

    Cullen DW, Toth IK, Pitkin Y, Boonham N et al. 2005 - Use of quantitative molecular diagnostic assays to investigates Fusarium dry rot in potato stocks and soil. Phytopathology 95, 1462-1471. doi: 10.1094/PHYTO-95-1462

    CrossRef    Google Scholar

    Dix NJ, Webster J. 1995 -Fungal ecology. Japanese Journal of Ecology 45, 332-333.

    Google Scholar

    Dubey RC, Dwivedi RS. 1986 -Destructive mycoparasitic behaviour of Fusarium solani (Mart. ) App. and Woll. against Mucor spinosus Van Tieghem. Microbios Letter 32, 123-127.

    Google Scholar

    Duffy B, Schouten A, Raaijmakers JM. 2003 -Pathogens cell defense: mechanisms to counteract microbial antagonisms. Annual Review Phytopathology 41, 501-538. doi: 10.1146/annurev.phyto.41.052002.095606

    CrossRef    Google Scholar

    El-Debaiky SA. 2017 -Antagonistic studies and hyphal interaction of the new antagonist Aspergillus piperis against some phytopathogenic fungi in-vitro in comparison with Trichoderma harzianum. Microbial Pathogenesis 113, 135-143. doi: 10.1016/j.micpath.2017.10.041

    CrossRef    Google Scholar

    Fakhrunnisa M, Hashmi H, Ghaffar A. 2006 -In vitro interaction of Fusarium spp. with other fungi. Parkish Journal of Botany 38, 1317-1322.

    Google Scholar

    Fernald M. 1970 - Gray's manual of botany: a hand book of the flowering plants and ferns of the central and northeast United States and adjacent Canada 8th edition. American Book Company, New York, 1251.

    Google Scholar

    Fones HN, Bebber DP, Chaloner TM, Kay WT et al. 2020-Threats to global food security from emerging fungal and oomycete crop pathogens. Nature Food 1, 332-342. doi: 10.1038/s43016-020-0075-0

    CrossRef    Google Scholar

    Fones H, Gurr S. 2015-The impact of Septoria tritici blotch disease on wheat: an EU perspective. Fungal Genetics Biology79, 3-7. doi: 10.1016/j.fgb.2015.04.004

    CrossRef    Google Scholar

    George PBL, Creer S, Griffiths RI, Emmett BA, Robinson DA, Jones DL. 2019 - Primer and data base choice affect fungal function but not biological diversity findings in a national soil survey. Frontiers in Environmental Science 7, 173. doi: 10.3389/fenvs.2019.00173

    CrossRef    Google Scholar

    Gibb GD, Strohl WR. 1987 - Physiological regulation of protease activity in Streptomyces peucetius. Canadian Journal of Microbiology 34, 187-190.

    Google Scholar

    Globe Newswire. 2018-Global fungicides market research report. https://www.globenewswire.com/en/news-release/2018/01/17/1295610/0/en/Global-Fungicides-Market-Noticeable-Shift-from-Inorganic-to-Organic-Fungicides-to-Propel-Growth-in-Market-finds-TMR.html (Accessed on May 15, 2021).

    Google Scholar

    Gomathi S, Ambikapathy V. 2011 - Antagonistic activity of fungi against Pythium debayanum (Hesse) isolated from chili field soil. Advances in Applied Science Research 2, 291-297.

    Google Scholar

    Hay WT, Fanta GF, Rich JO, Schisler DA, Selling GW. 2019 - Antifungal activity of a fatty ammonium chloride amylose inclusion complex against Fusarium sambucinum; control of dry rot on multiple potato varieties. American Journal of Potato Research 96, 79-85. doi: 10.1007/s12230-018-9683-8

    CrossRef    Google Scholar

    Hongsanan S, Sanchez S, Crous PW, Ariyawansa HA et al. 2016 -The evolution of fungal epiphytes. Mycosphere 7, 1690-1712. doi: 10.5943/mycosphere/7/11/6

    CrossRef    Google Scholar

    Ibrahim M, Tambuwal NI. 2015 -Mycoflora associated with post-harvest rots of tomato (Solanum lycopersiconL. ) fruits in Sokoto, Northwestern, Nigeria. Journal of Zoological and Bioscience 2(2), 42-46.

    Google Scholar

    Jayasiri SC, Hyde KD, Ariyawansa HA, Bhat J et al. 2015 - The Faces of Fungi database: fungal names linked with morphology, phylogeny and human impacts. Fungal Diversity 74(1): 3- 18. Doi10.1007/s13225-015-0351-8 doi: 10.1007/s13225-015-0351-8

    CrossRef    Google Scholar

    Jeffries P. 1995 -Biology and ecolgy of mycoparasitism. Canadian Journal of Botany 73, 1284- 1290. doi: 10.1139/b95-389

    CrossRef    Google Scholar

    Kredics L, Zsuzsanna A, Manczinger L, Szekeres A et al. 2003 - Influence of environmental parameters on Trichoderma strains with biocontrol potential. Food Technology Biotechnology 41, 37-42.

    Google Scholar

    Kumar D, Gouda A. 2018 - Evaluation of mycoparasitic efficacy of nematode-trapping fungi against Rhizoctonia solani inciting sheath blight disease in rice (Oryza sativa L. ). Science Direct 122, 31-40.

    Google Scholar

    Li KL. 1992 - The dry rot of potato in Hohhot. Acta Science National University Intramongolica23, 429-433.

    Google Scholar

    Matroudi S, Zamani MR, Motallebi M. 2009 - Antagonistic effect of three species of Trichoderma sp. on Sclerotinia sclerotiorum, the causal agent of canola stem rot. Egyptian Journal of Biology 11, 37-44.

    Google Scholar

    Nagano Y, Elborn JS, Millar BC, Goldsmith CE, Rendall J, Moore JE. 2008 - Development of novel PCR assay for the identification of the black yeast, Exophiala (Wangiella) dermatitidis from adult patients with cystic fibrosis (CF). Journal of Cystic Fibrosis 7, 576-580. doi: 10.1016/j.jcf.2008.05.004

    CrossRef    Google Scholar

    Okigbo RN, Ogbonnoya UO. 2006 -Antifungal effects of two tropical plant leaf extracts (Ocimum gratissimum and Aframomum melegueta) on postharvest yam (Dioscorea spp. ) rot. African Journal of Biotechnology 5, 721-731.

    Google Scholar

    Peng XW, Zhu JH. 2008 - Variety and distribution of potato fungi disease in Herbei Province. China Potato Journal 22, 31-33.

    Google Scholar

    Premalatha K, Gokul S, Amit K, Priyanka M et al. 2014 -Molecular profiling of fungal assemblages in the healthy and infected roots of Decalepis arayalpathra (J. Joseph & V. Chandras) Venter, an endemic and endangered ethnomedicinal plant from Western Ghats, India. Annals of Microbiology 65. doi: 10.1007/s13213-014-0919-7

    CrossRef    Google Scholar

    Raaijmakers J, Vlami M, De Souza J. 2002 - Antibiotic production by bacterial biocontrol agents. Kluwer Academic Publishers 81, 537-547.

    Google Scholar

    Shoresh M, Harman G, Mastouri F. 2010 -Induced systematic resistance and plant responses to fungal biocontrol agents. Annual Review of Phytopathology 48, 21-43. doi: 10.1146/annurev-phyto-073009-114450

    CrossRef    Google Scholar

    Spooner DM, Knapp S. 2013 - Solanum stipuloideum Rusby, the correct name for Solanum circaeifolium Bitter. American Journal Potato Research 90, 301-305. doi: 10.1007/s12230-013-9304-5

    CrossRef    Google Scholar

    Stefanczyk E, Sobkowiak SM, Brylinska M, Sliwka J. 2016 -Diversity of Fusarium spp. associated with dry rot of potato tubers in Poland. Europe Journal Plant Pathology 145, 871-884

    Google Scholar

    Verma M, Brar SK, Tyagi RD, Surampalli RY, Valero JR. 2007 -Antagonistic fungi, Trichoderma spp. : panoply of biological control. Biochemical Engineering Journal 37, 1-20. doi: 10.1016/j.bej.2007.05.012

    CrossRef    Google Scholar

    Waing KGD, Abella EA, Kalaw SP, Waing FP, Galvez CT. 2015 -Antagonistic interaction among different species of leaf litter fungi of Central Luzon State University. Plant Pathology & Quarantine 52, 122-130.

    Google Scholar

    Wharton P. 2015 - Potato disease Fusarium dry rot (E2992). Department of Plant Pathology, Michigan State University. https://www.canr.msu.edu/resources/potato_diseases_fusarium_dry_rot_e2992 (Accessed on November 15, 2018).

    Google Scholar

    Wharton P, Hammerschmidt R, Kirk W. 2007 -Department of Plant Pathology, Michigan State University. https://www.canr.msu.edu/uploads/files/fusarium-dry-rotbulletin.pdf (Accessed on November 15, 2018).

    Google Scholar

    Xing KY, Guo SX, Lee MW. 2005 -Morphological characteristics of hyphal interaction between Grifola umbellate and its companion fungus. Mycobiology Journal 33, 1-6. doi: 10.4489/MYCO.2005.33.1.001

    CrossRef    Google Scholar

    Ye Q, Wang, GC. 1994 - On Fusarium dry rot of potato in Zhejiang. Acta Phytopathology 5(14).

    Google Scholar

  • Cite this article

    GAD Carreon, EE Gandalera, KGD Waing. 2021. Molecular identification and in vitro interaction of molds associated with dry rot of potato (Solanum tuberosum L.) collected in La Trinidad, Benguet, Philippines. Studies in Fungi 6(1):315−326 doi: 10.5943/sif/6/1/22
    GAD Carreon, EE Gandalera, KGD Waing. 2021. Molecular identification and in vitro interaction of molds associated with dry rot of potato (Solanum tuberosum L.) collected in La Trinidad, Benguet, Philippines. Studies in Fungi 6(1):315−326 doi: 10.5943/sif/6/1/22

Figures(7)  /  Tables(2)

Article Metrics

Article views(4031) PDF downloads(572)

Other Articles By Authors

ARTICLE   Open Access    

Molecular identification and in vitro interaction of molds associated with dry rot of potato (Solanum tuberosum L.) collected in La Trinidad, Benguet, Philippines

Studies in Fungi  6 Article number: 22  (2021)  |  Cite this article

Abstract: Solanum tuberosum L. from the Solanaceae family is one of the most important agricultural crops grown in the Philippines next to rice. Unfortunately, harvested potatoes were plagued with various fungal diseases resulting to occurrence of dry rot affecting the production and harvest of potatoes. Dry rot in potatoes is caused by several fungal species in the genus Fusarium. Hence, this study aimed to isolate and identify fungal species associated with dry rot disease of potato. Potatoes showing diseased symptoms of dry rot were randomly selected and collected from various traders in La Trinidad, Benguet and were subjected for fungal isolation through serial dilution and plating. Identification was done through observation of cultural and morphological characteristics as well as DNA barcoding using ITS region. A total of six fungal species were subjected into BLAST which revealed the following species: Aspergillus flavus (100.00%), A. fumigatus (100.00%), A. niger (99.82%), Fusarium oxysporum (95.06%), F. solani (100.00%) and Mucor velutinosus (96.45%). To establish possible fungal species as potential biocontrol agent that would lessen the use of harmful chemicals, the identified fungal organisms were interacted with one another through in vitro interaction. Results showed that fungi exhibited antagonistic interaction against each other. Hyphal denaturation, hyphal penetration, hyphal coiling and cell lysis, were observed through microscopic observation. Meanwhile, M. velutinosus showed the ability to penetrate the host hyphae of the F. solani and F. oxysporum. Therefore, Mucor velutinosus could be a potential biological control agent against Fusarium species, which can reduce the use of harmful fungicides in controlling fungal diseases in potato that causes dry rot.

  • Potato (Solanum tuberosum L.), locally known as "patatas" is widely cultivated in 130 countries worldwide including the Philippines. According to Fernald (1970) and Spooner & Knapp (2013), Solanum species comprise more than 1000 species. Potato follows rice and wheat as one of the most important food crop for human consumption. The nutritional composition of potato has major impacts on population health such as the presence of vitamins and minerals thereby preventing malnutrition and slows down the accumulation of chronic diseases because of its antioxidant content (Camire et al. 2009).

    However, this staple food can be infected by post-harvest diseases caused by fungi such as dry rot, caused by several species of Fusarium causing great loss in crop production. Accordance to the earlier reports, there are 13 Fusarium species considered as causal agents of dry rot of potatoes worldwide (Cullen et al. 2005) and the most frequent and devastating are F. sambucinum, F. solani and F. oxysporum (Li 1992, Ye & Wang 1994, Peng & Zhu 2008, Hay et al. 2018). Infection starts when the pathogen enters the tuber, causing rotting out of the left, and once the pathogens penetrated the tuber skin, it begins to develop internally into the tissue (Wharton et al. 2007).

    To control fungal diseases, chemical fungicides are extensively used in agriculture but can cause problems such as environmental pollution and deterioration of human health (Gomathi & Ambikapathy 2011). Consequently, studies reveal that a fungi can be used as a biocontrol agent if interactions between other fungi can be determined (Duffy et al. 2003). Moreover, biological control agents have specific advantages over synthetic fungicides. It was proven to have effective control of pathogens and reduces pests with the use of natural enemies (Brimmer & Boland 2003, Adebola & Amadi 2010). Hence, the present study was conducted to isolate and identify molds associated with dry rot of potato. Interactions between the identified molds were carried out to determine the species of fungi that have potential as bio control agent.

  • Potato tubers showing diseased symptoms such as dark depressions on the surface, wrinkled skin in concentric rings, necrotic areas from light to dark brown or black in color and rotted cavities (Wharton 2015) were randomly selected and obtained from 5 traders of La Trinidad, Benguet Vegetable Trading Post. Tubers were kept in proper labeled paper bags and immediately transported to laboratory for isolation.

  • Collected potato tubers were rinsed with tap water to remove the dust and adhering debris. Then, the infected part of the sample was cut from the healthy part using sterilized blades under aseptic conditions. Infected part were then rinsed with sterile distilled water for 1 minute. Then, infected potato parts were then pulverized using a blender. Ten grams from the pulverized sample were added with 90 ml sterilized distilled water and was regarded as 10-1. Six test tubes were prepared having 9 ml of sterilized distilled water each and were labeled from 10-2 to 10-7. One ml from 10-1 dilution was transferred into 10-2 dilution and was shaken. Then, one ml from 10-2 was transferred to 10-3 dilution. This procedure was done in which one ml was being transferred from one dilution to another up to 10-7 dilution. Then, one ml of sample from 10-1, 10-3, 10-5 and 10-7 were poured on sterilized petri plate separately and pour plated with approximately 20 ml previously sterilized Potato Dextrose Agar (PDA). Triplicate was made for each plates. It was incubated at room temperature in alternating light and dark conditions for 4-5 days or until sufficient mycelial growth was available for isolation.

  • Distinct fungal colonies were purified into pure culture using three-point inoculation technique. Using a sterilized needle, mycelia were isolated from the mixed culture into a previously plated PDA. The plated isolates were incubated for 4-7 days at room temperature. Isolates were only considered as pure culture when colonies show consistent growth characteristics. Pure culture of the fungal isolates were deposited at the Biodiversity Conservation Laboratory, Department of Biological Sciences, College of Science, Central Luzon State University, Science City of Munoz, Nueva Ecija, Philippines, 3120. Facesoffungi numbers were registered as mentioned in Jayasiri et al. (2015).

  • Three-point inoculation was done in previously prepared plated PDA to observe the cultural characteristics of the six isolates. The re-growth, size and pigmentation of fungi were observed and recorded.

  • In a petri plate with moistened tissue paper, a clean glass slide was placed on top of a v-shaped foil and was sterilized. Agar block approximately 1 cm-thick was placed on the left of the slide. Fungal species were inoculated on the agar block and covered with sterilized cover slip. Inoculated slides were incubated for 3-5 days at room temperature. After incubation, morphological characteristics were observed under a compound microscope.

  • Seven-day old fungal isolates cultured on test tubes with PDA medium was sent to Philippine Genome Center in Quezon City, Manila for DNA extraction and sequencing following their standard procedures and protocols. In which DNA extraction was done by CTAB method using universal ITS1 (5'- TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') primers with 58'C annealing temperature for polymerase chain reaction. Capillary sequencing was carried out on the ABI 3730xl DNA Analyzer using a 50cm 96-capillary array, POP7TM Polymer, and 3730xl Data Collection Software v3.1 and base calling was done on Sequencing Analysis Software v5.4. Trimming and assembling of sequences were done using Codon Code Aligner V8.0.2

  • Two fungi were inoculated using one-point technique at the opposite sides of the previously plated PDA and then allowed to ramify at room temperature for seven days. After incubation, colony growth was observed and their interaction was determined (Waing et al. 2015).

  • Interactions of fungi were described as antagonism and mutual antagonism. Fungal isolates were classified as victim or aggressor in all antagonism tested (Dix & Webster 1995). Mutual inhibition or mutual slight inhibition is determined if there is mutual antagonism between fungi. In mutual inhibition, the fungus approached each other until almost contacted and a demarcation line of more than 2 mm was visible while mutual slight inhibition shows a distance of 0.1 to 2mm between two fungal colonies (Fakhrunnisa et al. 2006).

  • The procedure for interfungal parasitic relationship was adopted from Matroudi et al. (2009) with minor modifications, in which the slide culture technique determined the antagonistic interaction between fungal species. In a petri plate with moistened tissue paper, a clean glass slide was placed on v-shaped foil and was sterilized. The inocula of the two fungi were placed on both ends measuring 1 cm apart of sterilized glass slide separately. One end of the slide was coated with approximately 5.0 mm-thick layer of PDA while the other end was kept free for handling. The inoculated slides were incubated at room temperature for 3-5 days. After incubation, presence of coil formation and penetration structures, or wall disintegration where both hyphae meet were observed microscopically.

  • A total of six species of fungi were identified from infected potato with dry rot disease. The external part of diseased potato was seen (Fig. 1A) having dark depression on the surface with large lesions and the skin was dry and wrinkled (Fig. 1B). The internal diseased part of the potato was observed (Fig. 1C) with mycelial clumps of varying color from white to yellow to pink.

    Figure 1.  Observed symptoms of dry rot in samples collected in La Trinidad. A Benguet showing surface dark depression. B wrinkled and dry skin. C mycelial clumps in dead skin.

  • The cultural characteristics of isolated fungi described on the 7th day of incubation were as follows: A. flavus colonies (Fig. 2A) formed fastidious whitish mycelium at the margin and yellow green color at the left on obverse side of the plate. Mycelium at reverse side was dirty white in appearance with light brown color at the left with a diameter of 48.80 mm. Colonies of A. fumigatus (Fig. 2B) attained 58.18 mm in diameter. On its surface and margin, it appears blue green in color with white fluffy mycelium. Reverse side was dark blue to pinkish in appearance with light brown color at the left. The colonies of A. niger (Fig. 2C) in PDA plate produce white spreading mycelium at the margin with powdery pale yellow to black color mycelium on the left in both sides of the agar plate. Colony growth measures 51.15 mm. The colonies of F. oxysporum (Fig. 2D) were peach in color with velvetly aerial mycelium. Reversed side of the plate was yellowish to orange with dark violet mycelium on its left. Colony diameter was 57.29 mm. The colonies of F. solani (Fig. 2E) in obverse side of the plate were seen creamy white in appearance with cottony mycelium. Reversed side appears yellowish to pale brown mycelium, and colony diameter measures 46.87 mm. M. velutinosus (Fig. 2F) colonies at obverse side of the plate was seen powdery gray in color and white on the left. Reverse side of PDA plate show pale yellow in the left with white concentric rings. Colony diameter was 61.48 mm.

    Figure 2.  Cultural characteristics represented by A A. flavus. B A. fumigatus. C A. niger. D F. oxysporum. E F. solani. F M. velutinosus.

  • Observed morphological characteristics of the isolated fungi were described on the 7th day incubation were as follows: The stipes of A. flavus (Fig. 3A) appears rough and light brown, while the conidium was rough and have spherical surface. The conidia of A. fumigatus (Fig. 3B) is non septated and have globose shape with rough edges, conidiophores appears hyaline. Conidiophores of A. niger (Fig. 3C) are branched with lumped phialides. The conidiophores of F. oxysporum (Fig. 3D) were long with fusiform conidia, hypha is hyaline with branch monophialides. F. solani (Fig. 3E) shows narrow macro conidia, and long and branch monophialides. M. velutinosus (Fig. 3F) have globose sporangiospores, and formed septated hyphae.

    Figure 3.  Morphological characteristics represented by A A. flavus. B A. fumigatus. C A. niger.D F. oxysporum. E F. solani. F M. velutinosus (400X).

  • The identities of fungal organisms isolated from the S. tuberosum infected with dry rot disease were confirmed through amplification and sequencing of the Internal Transcribed Spacer region (ITS) region using ITS1 and ITS4 primers. BLAST analysis showed the identities of fungal isolates, namely Aspergillus flavus FoF 09597 (100%), Aspergillus fumigatus FoF 10086 (100%), Aspergillus niger FoF 10087 (99.82%), Fusarium oxysporum FoF 03824 (95.06%) Fusarium solani FoF 01873 (100%) and Mucor velutinosus FoF 10088 (96.45%) as shown in Table 1.

    Table 1.  Identities of the identified fungi with Facaesoffungi number using BLAST with NCBI Genbank Accession number.

    Isolate No. FacesofFungi Number Species E-Value Identify Accession
    1 FoF 09597 Aspergillus flavus 0.0 100.00% MN511747.1
    2 FoF 10086 Aspergillus fumigatus 0.0 100.00% MF540309.1
    3 FoF 10087 Aspergillus niger 0.0 99.82% KY607770.1
    4 FoF 03824 Fusarium oxysporum 0.0 95.06% MG372014.1
    5 FoF 01873 Fusarium solani 0.0 100.00% MN202790.1
    6 FoF 10088 Mucor velutinosus 0.0 96.45% KY203942.1
  • Meanwhile, antagonistic interaction was observed in all fungal isolates (Table 2). Antagonism in all fungal isolates was identified as aggressor or victim. The observed interfungal parasitic relationships were hyphal penetration, hyphal coiling, hyphal denaturation, hyphal branching and lysed cells, all observed under the compound microscope (Figs 4-7) (Waing et al. 2015).

    Table 2.  Antagonistic interaction of isolated fungi in potato tuber infected with dry rot disease.

    Interacting Fungi Interaction
    M. velutinosus(+) A. flavus(-) Antagonism
    M. velutinosus(+) A. fumigatus(-) Antagonism
    M. velutinosus(+) F. solani(-) Antagonism
    M. velutinosus(+) F. oxysporum(-) Antagonism
    M. velutinosus(-) A. niger(+) Antagonism
    A. flavus(-) A. fumigatus(+) Antagonism
    A. flavus(+) F. solani(-) Antagonism
    A. flavus(-) F. oxysporum(+) Antagonism
    A. flavus(-) A. niger(+) Antagonism
    A. fumigatus(-) F. solani(+) Antagonism
    A. fumigatus(-) F. oxysporum(+) Antagonism
    A. fumigatus(-) A. niger(+) Antagonism
    F. solani(-) F. oxysporum(+) Antagonism
    F. solani(-) A. niger(-) Antagonism
    F. oxysporum F. oxysporum Antagonism
    F. oxysporum(+) A. niger(-) Antagonism
    (+) aggressor, (-) victim

    Figure 4.  Antagonistic interaction represented by; A M. velutinosus (left) and A. flavus (right). B hyphal denaturation and lysed cells of A. flavus (400x). C M. velutinosus (left) and A. fumigatus(right). D hyphal denaturation and lysed cells of A. fumigatus (400x). E M. velutinosus (left) and F. solani (right). F hyphal penetration of M. velutinosus to F. solani (400x). G M. velutinosus (left) and F. oxysporum (right). H hyphal penetration of M. velutinosus to F. oxysporum (400x). I M. velutinosus (left) and A. niger (right). J hyphal denaturation and lysed cells of M. velutinosus (400x).

    Figure 5.  Antagonistic interaction represented by; A A. flavus (left) and A. fumigatus (left). B lysed cells of conidiophores of A. flavus (400x). C A. flavus (left) and F. oxysporum (right). D hyphal coiling of F. oxysporum leading to hyphal denaturation of A. flavus (400x). E A. flavus (left) and A. niger (right). F hyphal denaturation of A. flavus (400x). G A. flavus (left) and F. solani (right). H hyphal denaturation and lysed cells of F. solani (400x).

    Figure 6.  Antagonistic interaction represented by; A A. fumigatus (left) and F. solani (right).B hyphal denaturation of A. fumigatus (400x). CA. fumigatus (left) and F. oxysporum (right). D lysed cells of A. fumigatus (400x). EA. fumigatus (left) and A. niger (right). F lysed cells of A. fumigatus (400x).

    Figure 7.  Antagonistic interaction represented by; A F. solani (left) and F. oxysporum (right). B hyphal penetration of F. oxysporum to F. solani (400x). C F. solani (left) and A. niger (right). D hyphal denaturation and lysed cell of F. solani (400x). E F. oxysporum (right) and A. niger (left). F hyphal penetration of F. oxysporum to A. niger (400x).

  • Observed characteristics of infected potato with dry rot disease are similar to the study of Wharton (2015). In addition, wounds caused by unavoidable process of harvesting potato allowed the manifestation of fungal organisms that later on led to dry rot disease (Wharton et al. 2007). The present study has isolated two species of Fusarium, F. solani and F. oxysporum in which based on the study conducted by Stefanczyk et al. (2016), Fusarium species have been associated with potato dry rot. Such, F. sambucinum, F. solani and F. oxysporum are the most frequent and devastating species. In addition, three species of Aspergillus namely: A. flavus, A. fumigatus and A. niger were also isolated. According to the study of Ibrahim & Tambuwal (2015), A. niger, and A. fumigatus were both pathogenic causing rotting of tomato fruit. Meanwhile, A. flavus was previously isolated from Dioscorea spp. rot (Okigbo & Ogbonnaya 2006). Moreover, M. velutinosus is one of the most frequent fungal endophyte identified from the rotted root samples of D. arayalpathra (Premalatha et al. 2014). Fungal epiphytes are found on the surface of the plants. Also, many of them are obligate parasites which penetrated the host plant and prevent the uptake of the nutrients as cited by Hongsanan et al. (2016). These isolated fungal species are confirmed through BLAST analysis. In which based on the previous study of Nagano et al. (2008) the use of ITS primer for fungal identification shows accurate and reliable result. Furthermore, ITS primer is the standard marker for fungal DNA barcoding (Bellemain et al. 2010) and is generally used for the assessment and characterization of fungal communities from environmental samples (George et al. 2019).

    The interaction study revealed antagonism between all identified fungi. Hyphal denaturation, hyphal coiling, hyphal penetration and lysed cells were the observed antagonistic interaction of hypha. In which, M. velutinosus showed the ability to penetrate the host hyphae of the F. solani and F. oxysporum which are considered as causative agents of potato dry rot. In microbial communities, there are different relationships and interactions characterized by mutualism to antagonism and parasitism (Duffy et al. 2003). These interactions happen when nutrients and space are limited (Arya & Perello 2010). Fungi that affects the other by harming them by means of suppressing the growth of other or killing them is mycoparasitism or hyperparasites (Jeffries 1995, Kumar & Gouda 2018). Its behavior which was observed in previous studies includes hyphal penetration, hyphal coiling, hyphal branching, hyphal denaturation, cell lysis, spore production, and barrier formation (Dubey & Dwivedi 1986, El-Debaiky 2017). As a result of these interactions, the host hyphae were vacuolated, shrank, collapsed, and disintegrated. Moreover, at the interface region of colonies, certain fungi were observed having irregular branched hyphae, cracked hyphae, ruptured cell wall, and dead cells at the left of antagonism line (Chet et al. 1981, Xing et al. 2005). The ability of mycoparasites to penetrate the host cell after coiling can lead to cell lysis because of the enzymatic interaction. Wherein, certain fungus penetrates internally and uses direct enzymatic lysis of hyphal cells. Hyphal coiling incorporated by means of hock between the hyphae and lysed cells can lead to fusion. In addition, hyphal cell wall lysis leads to penetration of the hyphae for obtaining nourishment to the host by means of haustoria (Dubey & Dwivede 1986, El-Debaiky 2017). However, certain host fungi counter attack by forming resting bodies. Also, host hypha accumulates cytoplasm to stop the infiltration of antagonist inside the cell. These host defense act as a barrier and survival measures against destruction in adverse condition and the over expression of pyridoxal reductase is a result of stress condition of a certain host fungi (Dubey & Dwivede 1986, Chamoun & Jabaji 2011, El-Debaiky 2017). According to the studies of Barnette (1963) and El-Debaiky (2017), the degree of damage by the parasites to the host cells depends to the resistance of host species. These studies show that certain parasites have direct contact to the host because of the absence of diffusible antibiotics which caused severe damage. In addition, some antagonist can produce spore inside the host hypha depending to the nutrition from the host fungi. Furthermore, according to Verma et al. (2007), there are several modes of actions that mycoparasitism exhibited which includes competition in space and nutrient, production of antibiosis and plant defense system induction. One evidence of this mode was observed by Waing et al. (2015) in which, the presence of zone of inhibition was observed when the two fungi are paired which indicates the production of antibiotics.

    Emerging fungal diseases in various agricultural crops due to rapid growth of new pathogens that are fungicide resistant posed a significant threat in agricultural productivity and risk to global food security (Fones et al. 2020). An example of which is the fungal disease known as Septoria tritici blotch (STB) that resulted to million per year in yield losses in UK growers alone (Fones & Gurr 2015). In order prevent loss due to fungal pathogens, fungicides were being applied. However, commercially available fungicides are expensive and became an added burden to farmers at the same time it is not environmental friendly (Gomathi & Ambikapathy 2011, Globe Newswire 2018). Thus, public concerns arise due to the hazardous effect of these harmful chemicals in the field of agriculture. Therefore, biocontrol microorganism has been stimulated as a substitute and answers for the pest control (Raaijmakers et al. 2002) and biocontrol pf pathogens. Several mycoparasites which belongs to the filamentous fungal genus were the best candidates for bio control agent against fungal pathogens (Kredics et al. 2003). Species exhibited extracellular enzyme such as polygalaturonase, amylase, protease and lipase which enable them to absorb nutrients (Gibb & Strohl 1987, Alvez et al. 2002). Thus, certain biocontrol agents not only function as pest control but can also enhance the uptake of nutrients in plants, slow down the occurrence of abiotic stresses and some can reduce physiological stresses (Shoresh et al. 2010).

Figure (7)  Table (2) References (45)
  • About this article
    Cite this article
    GAD Carreon, EE Gandalera, KGD Waing. 2021. Molecular identification and in vitro interaction of molds associated with dry rot of potato (Solanum tuberosum L.) collected in La Trinidad, Benguet, Philippines. Studies in Fungi 6(1):315−326 doi: 10.5943/sif/6/1/22
    GAD Carreon, EE Gandalera, KGD Waing. 2021. Molecular identification and in vitro interaction of molds associated with dry rot of potato (Solanum tuberosum L.) collected in La Trinidad, Benguet, Philippines. Studies in Fungi 6(1):315−326 doi: 10.5943/sif/6/1/22
  • Catalog

    • About this article

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return