Wikee S; Chumnunti P; Kanghae A; Chukeatirote E; Lumyong S; Faulds CB

Research Article

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

Lignocellulolytic Capability of Endophytic Phyllosticta sp.

Wikee S¹, Chumnunti P^2,3, 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

Abstract

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

Introduction

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).

Table 1: Chemical composition of lignocellulosic biomass used as growth substrates.




  
    Substrates
    Carbohydrate
    lignin
    protein
    fiber
  
  
    cellulose
    hemicellulose 
    arabinose
    xylose
    galactose
    mannose
    glucose
    rhamnose
  
  
    Ricestraw [52-53] 
    32.5 
    28 
    3.3±0.2 
    20.7±0.2 
    1.2±0.2 
    0.5±0.2 
    41.7±2.2 
    0.3±0 
    19.64 
    7.9±2.4 
    20-40 
  
  
    Ricehusk [54-56] 
    25-35 
    18-21 
    1.7 
    14 
    1.6 
    0.3 
    33.4 
    0.1 
    26-31 
    2.9-3.6 
    39-42 
  
  
    Wheatstraw [57-59] 
    38 
    29 
    3.9±0.2 
    0.5±1.1 
    5.6-10.4 
    <1 
    36.3±1.8 
    0.3 
    15 
    4 
    32 
  
  
    Miscanthus [60-61] 
    43 
    24 
    1.1 
    14.9 
    0.3 
    0-14 
    38 
    0-1.6 
    19 
    3 
    6.7 
  
  
    Sorghum [62-63] 
    34-44 
    - 
    - 
    24 
    
    - 
    4.9 
    - 
    18-20 
    9.7 
    36.67 
  
  
    Lavanderstraw [64] 
    43±1.7 
    13±0.9 
    3.4±0.1 
    29±0.2 
    3 
    2.5 
    45.6 
    0.93 
    23±3.6 
    - 
    - 
  
  
    Lavanderflower [64] 
    22.4±2 
    12.6±2 
    7.3 
    17.9±0.1 
    6.2±0.2 
    3±0.1 
    36.2±0.4
    1.15 
    23.6±2.6 
    6.34 
    8.94



Table 1:  Chemical composition of lignocellulosic biomass used as growth substrates.

Table 2: Radial growth rate of the fungal strains cultivated in different biomass substrates.




  
    Fungal strains (MFLUCC) 
    Radial growth rate    (cm/day) 
  
  
    Miscanthus
    Rice husk
    Rice straw
    Sorghum
    Wheat Straw
    Lavender flower
    Lavender straw
    Glucose
  
  
    10-0137
    0.06
    0.05
    0.04
    0.17
    0.05
    0.06
    0.06
    0.07
  
  
    12-0015
    0.34
    0.36
    0.24
    0.27
    0.22
    0.31
    0.31
    0.22
  
  
    12-0232 
    0.34
    0.33
    0.31
    0.29
    0.23
    0.33
    0.33
    0.20
  
  
    14-0233 
    0.34
    0.33
    0.31
    0.24
    0.21
    0.27
    0.26
    0.26



Table 2:  Radial growth rate of the fungal strains cultivated in different biomass substrates.

Table 3: Enzyme activities were detected after recovered extracts and concentrated through a 10 kD membrane.




  
     
      Enzyme 
    Lavender flower 
    Lavender straw 
  
  
    protein(total mg) 
    Activity (U/mg) 
    protein (total mg) 
    Activity (U/mg) 
  
  
    Arabinase 
    1.8 
    53,287
    3.5
    45,211
  
  
    Cellulose
    1.8
    2,710
    3.5
    1,144
  
  
    Laccase
    1.8
    336
    5.8
    1,189
  
  
    Pectinase
    1.8
    13,238
    11.0
    31,731
  
  
    Xylanase
    1.8
    955
    3.5
    362



Table 3:  Enzyme activities were detected after recovered extracts and
concentrated through a 10 kD membrane.

Colonies of the phytopathogen P. citrimaxima (MFLUCC 10- 0137) were flat, initially white with abundant mycelium, gradually becoming greenish to dark green after 2–3 days growth, with white hyphae at the margin, eventually turning black. Very slow growing ( x =0.1 cm/day) in all treatments except sorghum ( x =0.2 cm/day). Between days 3-7, Phyllosticta citrimaxima was fast growing on all substrates, stabilizing after 7 days, except for growth on sorghum, where the diameter of growth reached 4.25 cm after 20 days. The colonies of all the endophyte P.capitalensis were flat, initially greenish to dark green after 2–3 days, eventually turning black. Fast growing (between =0.3 and 0.35 cm/day) on all substrates, with diameter of growth of P. capitalensis MFLUCC12-015 reaching 8.0 cm on rice husk on day 14. On day 3-7, P.capitalensis MFLUCC12-015 was fast growing in every growth conditions at day 2-10, but the growths were stable after 10 days. Between day 5-7, P. capitalensis MFLUCC12-0232 started to producing conidia on sorghum biomass agar. P. capitalensis (MFLUCC 14-0233), start producing conidia on sorghum biomass agar and pycnidia could be observed on biomass residue after day 5-7 of growth (Figure 1).

Figure 1: Growth of the four Phyllosticta sp. on different biomass mass media (15% w/w).

    
    
    Figure 1: Growth of the four Phyllosticta sp. on different biomass mass media
(15% w/w).

The rate of growth on lavender flowers and straw was initially faster for all species, except for P. citrimaxima MFLUCC10-0137, especially over the first 6 days. At day 3, the growing edge of P. capitalensis MFLUCC12-015 mycelium reached almost 3 cm, compared to growth on the other substrates, where it reached only 0.8-1.8 cm over the same time period (Figure 2). Growth tended to stabilize after 10 days. On lavender flower and lavender stem biomass agar, the agar at the margin of the leading edge of most fungal strains changed to a red color during 1-3 days of incubation and turn darker after 7-20 days, except for P. citrimaxima. Most of strains could produce pycnidia and conidia on biomass residue (Figure 3) except for P. citrimaxima. Essential oils from plants, such as lavender, oregano, sage and mint are generally conceived as inhibitory to fungal growth [43]. This result suggests that in the residual biomass after oil extraction may contain compound(s) which are stimulatory to growth and thus opens the possibility of using lavender by-products for biomass treatment processes within a bio refinery concept.

Figure 2: Radial growth rate of the fungal strains cultivated on different biomasses substrates (a) Phyllosticta citrimaxima (MFLUCC10-0137); (b) Phyllosticta capitalensis (MFLUCC12-0015); (c) Phyllosticta capitalensis (MFLUCC12-0232); and (d) Phyllosticta capitalensis (MFLUCC 14-0233) on biomass mass media.

    
    
    Figure 2: Radial growth rate of the fungal strains cultivated on different
biomasses substrates (a) Phyllosticta citrimaxima (MFLUCC10-0137); (b)
Phyllosticta capitalensis (MFLUCC12-0015); (c) Phyllosticta capitalensis
(MFLUCC12-0232); and (d) Phyllosticta capitalensis (MFLUCC 14-0233) on
biomass mass media.

Figure 3: Represent characteristics of Phyllosticta capitalensis (MFLUCC14-0233) on sorghum media agar. A. Colony on Sorghum media agar B. Production of spore on media C-D. Sexual stage (Guignardia) E. Appressoria and germ tube F. Conidiophores G. Conidia with apical appendage (asexual stage).

    
    
    Figure 3: Represent characteristics of Phyllosticta capitalensis
(MFLUCC14-0233) on sorghum media agar. A. Colony on Sorghum media
agar B. Production of spore on media C-D. Sexual stage (Guignardia)
E. Appressoria and germ tube F. Conidiophores G. Conidia with apical
appendage (asexual stage).

Growth profiles are thought to identify differences between fungal species and strains which could be correlated to their genetic enzyme compliments and expression patterns. While two Dothideomycetes with different life styles show differences during growth on Dothistroma minimal media, C. fulvus and D. septosporum showed similar growth on simple sugars (25 mM), birchwood xylan, apple pectin and lignin (1% w/v) over 2-4 weeks, although the later grew slightly better on pectin, contradictory to its lower pectinolytic gene number but supported by the number of pectinolytic enzymes expressed during growth on their respective natural plant hosts [44]. This suggests that the expression regulation of gene involved in plant deconstruction is a more dominant factor than the number of the lignocellulosic genes in the genomes.

Enzyme activities of the Phyllosticta sp. during growth on the lignocellulosic biomass

The culture supernatants produced during growth of the four Phyllosticta strains on the 7 lignocellulosic residues were assayed to determine lignocellulose-degrading enzyme activities as well as the total amount of protein and total reducing sugars released into the media. All fungal supernatant from lavender media showed high amount of total protein; however, the activity levels were very low. All reducing sugar appeared to be consumed at the end of cultivation of P.capitalensis MFLUCC12-0015 and MFLUCC12-0232 while the level of reducing sugars increased during 5-7 days of cultivation of P. citrimaxima MFLUCC10-0137 and P. capitalensis MFLUCC14-0233. No cellulase, xylanase, peroxidase, esterase or arabinofuranosidase activity could be detected. Either the fungi do not produce these enzymes or the levels are too dilute in the sample extracts for detection, even after concentration.

P. capitalensis MFLUCC14-0233 growths

Due to the more rapid growth of P. capitalensis MFLUCC14-0233, the fungus was chosen to be cultured on Lavender Flower Medium (LFM), Lavender Straw Medium (LSM), Wheat Straw Medium (WSM) and potato Dextrose Broth (PDB). During the days of incubation, extracellular protein from LFM was determined to be 1.76±0.75 mg, 1.81±0.23 mg, and 12.89±1.35 mg at days 5, 7 and 10, respectively, while extracellular protein on LSM was determined to be 3.49±1.51 mg protein, 0.83±0.05 mg protein, 10.95±1.38 mg protein, and 5.76±1.19 mg protein at day 3, 5, 7, and 10, respectively. Extracellular protein was no longer detected at day 12. Additionally, extracellular protein in the supernatants from WSM and PDB grown cultures could also not be detected. A small amount of protein was detected in the supernatant of LSM at day 3, dropping by day 5 and reaching the highest concentration on day 7. On the other hand, protein in LFM could not be detected at day 3, increasing after day 5 and 7 then reached maximum protein concentration at day 10. Protein levels rapidly decreased to zero at day 12 during growth on both lavender media. Reducing sugar release, as glucose equivalents, was monitored at day 3, 5, 7, 10, and 12. The highest concentration of reducing sugar could be detected on day 3 for LFM (7.36±0.86 mM) and rapidly decreased after two days. The sugar concentration in the culture supernatant from LSM at day 3 was 3.86±0.44 mM, dropping to 1.93±0.69 mM at day 12. P.capitalensis cultured on LSM and LFM for 12 days and selected enzyme activities were determined.

Pectinolytic activity

The maximum production of arabinasein LSM occurred after 3 days of incubation (904.22 U/ml) while arabinose activity in LFM could not be detected. However, the maximun activity of arabinase produced during growth on LFM was 944.18 U/ml at day 10. Pectinase activity in concentrated extracellular supernatant of P. capitalensis in LFM showed a maximum activity against apple pectin of 448.39 U/ml at day 7 and 465.47 U/ml at day 10. In comparison, activity against citrus pectin was lower, with the highest activity at day 7 in LFM (263.37 U/ml) then at day 10 for LSM (245.11 U/ml) (Figure 5).

Download PDF

Citation: Wikee S, Chumnunti P, Kanghae A, Chukeatirote E, Lumyong S and Faulds CB. Lignocellulolytic Capability of Endophytic Phyllosticta sp.. J Bacteriol Mycol. 2017; 4(2): 1047. ISSN : 2471-0172

Instruction for Authors

Submit Your Article

Enzyme	Lavender flower		Lavender straw
Enzyme	protein(total mg)	Activity (U/mg)	protein (total mg)	Activity (U/mg)
Arabinase	1.8	53,287	3.5	45,211
Cellulose	1.8	2,710	3.5	1,144
Laccase	1.8	336	5.8	1,189
Pectinase	1.8	13,238	11.0	31,731
Xylanase	1.8	955	3.5	362