Lignocellulolytic Capability of Endophytic Phyllosticta sp.

Research Article

J Bacteriol Mycol. 2017; 4(2): 1047.

Lignocellulolytic Capability of Endophytic Phyllosticta sp.

Wikee S¹, Chumnunti P2,3, Kanghae A2, Chukeatirote E², Lumyong S¹ and Faulds CB4*

¹Department of Biology, Faculty of Science, Chiang Mai University, Thailand

²School of Science, Mae Fah Luang University, Chiang Rai, Thailand

³Institute of Excellence in Fungal Research, School of Science, Mae Fah Luang University, Chiang Rai, Thailand

4Aix Marseille Universite’, INRA, Biodiversité et Biotechnologie Fongiques (BBF), Marseille, France

*Corresponding author: Faulds CB, Aix Marseille Universite’, INRA, Biodiversité et Biotechnologie Fongiques (BBF), Marseille, France

Received: February 16, 2017; Accepted: March 22, 2017; Published: March 28, 2017


The Dothideomycetes represent the largest fungal class of Ascomycota. It is an ubiquitous class of fungi whose members span a wide spectrum of lifestyles and host interactions. The endophytic fungus Phyllosticta is one members of the Dothideomycetes, causing disease in economic crops. Phyllosticta was screened for the degradation of lignocellulosic biomass of commercial relevance, such as rice straw, rice husk, sorghum, wheat straw, miscanthus, lavender flower, and lavender straw. The highest degrading strains were identified from an initial screen and further analyzed for the secretion of lignocellulosic enzymes during growth on the different biomasses. With Phyllosticta capitalensis (MFLUCC14-0233), maximum activity of arabinase (944.18 U/ml culture), cellulase (27.10 U/ml), xylanase (10.85 U/ml), pectinase (465.47 U/ml), and laccase (35.68 U/ml) activities could be detected in the secretome during growth on lavender flowers and lavender straw. Phyllosticta capitalensis is thus an interesting new strain for the production of lignocellulosic enzymes during growth on cheap agro-industrial biomass.

Keywords: Agro-industrial residues; Biological pretreatment; Dothideomycetes; Lignocellulosic biomass; Lignocellulytic enzyme; Phyllosticta


The Dothideomycetes represent the largest fungal class within the phylum Ascomycota. It is a ubiquitous class of fungi whose members span a wide spectrum of lifestyles and host interactions [1- 3]. Members of the Dothideomycetes can cause disease in every major crop [4]. Approximately 1,300 genera and 19,000 species have been identified either as endophytes, plant pathogens, or as saprophytes degrading plant biomass, thus threatening agriculture and food security throughout the world [5-8]. In addition to their mode of life, the Dothideomycetes are known for producing secondary metabolites grouped into four main categories based on their biosynthetic origin: polyketides, non-ribosomal peptides, terpenoids and tryptophan derivatives [9]. These secondary metabolites can be both toxic and beneficial to plants and humankind in applications such as agrochemicals, antibiotics, immunosuppressant’s, antiparasitics, antioxidants and anticancer agents [10]. New Dothideomycetes are still being discovered worldwide and a large number of these strains remained unexplored regarding their potential use in biotechnology [11-12].

Endophytes provide a broad variety of bioactive secondary metabolites with unique structures and so this class of fungi could be used as potential “nanofactories” producing a range of “green” alternatives to currently employed chemicals [13]. Although the ecological significance of endophytes is not completely clear, it is known that these fungi can exploit dead leaves immediately after their senescence and before they fall from the tree [14], and so could be also exploited for the production of enzymes acting on plant biomass.

A well-known representative of Dothideomycetes fungi in metabolite studies is Phyllosticta (with a Guignardia anamorph). Phyllosticta species are mostly plant pathogens of a broad range of hosts and they are responsible for numerous diseases including leaf spot and black spots to spot on fruits. For example, P. ampelicida species causes black rot disease on grapevines [15], P. musarum species causes banana freckle disease [16], P. citricarpa is the cause of black spot on citrus and is regarded as a quarantine pest in Europe and the USA [17], and P. capitalensis while non-pathogenic, is found also on citrus usually isolated from black spot lesions and is known as an endophyte on an extensive range of host plants [6,18]. P. cartagena and P. ericae have been reported as saprophytes [19].

Phyllosticta spp. are commonly known to produce various kinds of secondary metabolites for example, Phyllosticta derivatives exhibiting growth-inhibitory activity in five cancer cell lines have been isolated from P. cirsii. Phytotoxins, phyllosinol, brefeldin, and PMtoxin are known as fungal pathogenic derivatives from Phyllosticta [20]. Recently, the phytotoxins guignarenones A-F and alaguignardic acid have been isolated which could stimulate the development of herbicides of natural origin [21-23]. In addition, antimicrobial activity active on growth inhibitor of Escherichia coli, Bacillus cereus, and Pseudomonas aeruginosa [24-25]. P. cirsii has been isolated from diseased leaves of Cirsium arvense and it evaluated as a potential biocontrol agent of this noxious perennial weed; furthermore, it produces different phytotoxic metabolites with potential herbicidal activity when grown in liquid cultures [20].

For any fungus which attempts to colonize a higher plant, whether it is endophytic, pathogenic, saprotrophic, biotrophic/necrotrophic, it must contend with the physical barriers of the host. There is a dearth of information however on the cell wall-acting enzymes produced by Phyllosticta sp. to help in the colonization of the leaves when compared to other endophytic and pathogenic fungi, such as Botryosphaeria sp. [26], Colletotrichum sp. [27], Fusarium sp. [28],

Macrophomina phaseolina [29], Magnaporthe sp. [30-31], smut fungi, such as Sporisorium scitamineum [32] and Ustilago maydis [33], and Stagonospora nodorum [34].

In this study, we examined the production of ligninolytic enzymes on seven previously characterized lignocellulosic agro-industrial residues by four Phyllosticta sp.: the pathogenic P. citrimaxima and three strains of the non-pathogenic endophyte P. capitalensis.

Materials and Methods

Collection of fungal cultures

Fungal cultures were obtained from Mae Fah Luang University Culture Collection (MFLUCC): the pathogen strains Phyllosticta citrimaxima MFLUCC10-0137 was isolated from Citrus maxima, P. capitalensis MFLUCC12-0015 and MFLUCC12-0232 were isolated from Euphorbia milii and Philodendron X ‘Xanadu’, respectively. An endophyte strain P. capitalensis MFLUCC14-0233 was isolated from Hevea brasiliensis [7]. Wheat straw and miscanthus were obtained from Vivescia (Reims, France), dried, and chopped (≈4 mm). Lavender straw and flowers were obtained as the residues after steam distillation from la société Bontoux SA (Saint Aubansurl’Ouveze, Drôme, France). They were collected at the beginning of September 2013, air dried for 12 days, flowers and straw separated then the fractions knife milled. Rice straw, husks and sorghum were obtained from rice cultivation fields (Phan, Thailand).

Growth measurement

Four strains of Phyllosticta were monitored on agar plates containing 15mg/ml biomass for 20 days in the different biomass media as follow; lavender flower (LF), lavender straw (LS), miscanthus (MC), rice straw (RS), rice husk (RH), sorghum (SG), wheat straw (WS). Growth was established by measuring the diameter of the growing edge of the mycelium with time. The growth measurement was recorded at day 3, 5, 7, 14, and day 20.

Preparation of fungal supernatant for enzyme essays

Growth condition in Liquid State Fermentation (LSF): The cultures were grown over 10 days in the presence of the seven biomasses in liquid medium (20 g/L), and stored at 30°C 130 rpm. The culture supernatants from all treatments were collected on days 0, 3, 7, 10, and 12 of incubation and stored at -20°C until use. The culture supernatants were then concentrated by filtration using a 0.2- micron-pore-size (polyethersulfone membrane; Vivaspin; Sartorius, Germany), diafiltered, and concentrated (Vivaspin polyethersulfone membrane with a 10-kDa cutoff; Sartorius) in 50 mM acetate solution buffer, pH5, and stored at -20°C until use [35].

Enzyme activities

The culture supernatants produced by the four Phyllosticta strains on different lignocellulosic substrates were assayed to determine the enzyme activities present. All chemicals were of the highest purity grade available and were purchased from Sigma-Aldrich (Saint-Quentin- Fallavier, France) if not stated otherwise. Cellulose degradation was assessed by the quantification of endo-1,4-β-D-glucanase (Azo- CM-Cellulose) (Megazyme, Ireland) and β-glucosidase (pNP-β-Dglucopyranoside [pGlu]) activities. Hemicellulose degradation was determined by quantifying the activity of endo-1, 4-β-D-Xylanase on azo-birchwoodxylan (Megazyme, Ireland), acetyl esterase on pNPacetate (pAc), and arabinose was assessed with 1% arabinogalactan [35,36]. Oxidative enzymes were assayed with ABTS (2,2’-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) [37] as a substrate for laccase activity, Manganese Peroxidase (MnP) by the oxidation of MnSO4 [38], and Lignin Peroxidase (LiP) was assayed using veratryl alcohol as substrate in the presence of H2O2 [39]. Pectinase activity was estimated by using apple pectin and citrus pectin [40]. The total amount of protein present in the culture supernatant was estimated using the Bradford assay with a BSA (Bovine serum albumin) standard curve [41]. Total reducing sugars were quantified using dinitrosalicylic acid method (DNS) [42]. All essays were performed in triplicate.

Results and Discussions

Growth profiles on the different lignocellulosic biomass

In this study, we focused on lignocellulosic residues from agroagriculture in Thailand and France such as lavender flower, lavender straw, miscanthus, rice straw, rice husk, sorghum, and wheat straw. Their chemical composition is shown in Table1. Growth of the four pure Phyllosticta strains was monitored on agar plates containing 15mg/ml biomass for 20 days. Growth was established by measuring the diameter of the growing edge of the mycelium over time (Table 2).