J. Gen. Appl. Microbiol., 50, 229–234 (2004)

Short Communication

Are terrestrial Ascomycetes lacking in alginolytic activity ?

Sabrina Sarrocco, Stefano Fanti, and Giovanni Vannacci*

Department of Tree Science, Entomology and Plant Pathology “G. Scaramuzzi,” Plant Pathology Section, Faculty of Agriculture, University of Pisa, Via del Borghetto 80, 56124 Pisa, Italy

(Received July 30, 2003; Accepted July 2, 2004)

Key Words——alginate; ; viscometric assay

Alginate is a gelling polysaccharide found in great rine fungi such as Corollospora intermedia, Aster- abundance as part of the cell wall and intracellular ma- omyces cruciatus and two species of Dendryphiella terial in the brown seaweeds (Pheophyceae). Most of salina, even if in these fungi production of alginolytic the alginates used commercially are obtained from enzymes seems an isolate-specific rather than a three genera, Macrocystis, Laminaria, and Ascophyl- species-specific trait (Schaumann and Weide, 1990; lum (Skjå´k-Braek and Martinsen, 1991) but alginate is Wainwright and Sherbrock-Cox, 1981). Numerous also produced by two families of heterotrophic bacte- methods have been developed for the detection of al- ria, the Pseudomonadaceae and Azotobacteraceae. ginolytic activity and the quantification of alginases. In- Alginates are (1, 4) linked glycuronans composed of corporation of alginate into growth media allows the residues of b-D-mannuronate (M) and its C5 epimer, a- screening of alginase-producing microorganisms, al- L-guluronate (G), arranged in block structures which though the most sensitive but also the most time-con- may be homopolymeric [polymannuronate (poly M) or suming method to quantify alginolytic activity is the vis- polyguluronate (poly G)] or heteropolymeric, often oc- cometric assay (Gacesa and Wusteman, 1990; Steven curring as random sequences (poly MG) (Haug et al., and Lewin, 1976). 1967). The susceptibility of alginates to degradation is No bibliographic references are available about the determined by both the block structure and degree of ability of fungal strains isolated from terrestrial environ- O-acetylation within the macromolecule. Alginate ment to utilize alginate as a carbon source. This study lyases catalyze the degradation of alginate by a b- aims at screening a number of terrestrial Ascomycetes elimination mechanism that has yet to be fully eluci- belonging to the fungal collection of this department for dated (Wong et al., 2000). Alginate degrading en- the ability to grow on alginate as sole carbon source zymes have been found in a variety of sources includ- and to quantify the ability to degrade alginate using a ing brown algae, molluscs (Madgwick et al., 1973, viscometric method. Alginate lyases have many appli- 1978; Watabane and Nisizawa, 1982), many genera of cations (Wong et al., 2000). Alginate oligomers display often unidentified marine and soil bacteria (Doubet and a large range of physiological activities both in humans Quatrano, 1984; Kaneko et al., 1990) and some ma- and plants (Iwamoto et al., 2003; Xu et al., 2003) and can be exploited to deliver anticancer agents (Bouhadir et al., 2001) or to induce the production of * Address reprint requests to: Prof. Giovanni Vannacci, De- partment of Tree Science, Entomology and Plant Pathology “G. useful secondary metabolites from plants (Akimoto et Scaramuzzi,” Plant Pathology Section, Faculty of Agriculture, al., 1999). The availability of unusual alginolytic en- University of Pisa, Via del Borghetto 80, 56124 Pisa, Italy. zymes, therefore, seems of reasonable scientific and E-mail: [email protected] industrial interest. 230 SARROCCO, FANTI, and VANNACCI Vol. 50

Table 1. Terrestrial fungal species evaluated for their ability to grow in presence of alginate as only carbon source.

Family Genusa Species No. tested isolates

Ascomycota, Ascomycetes Pleosporaceae Alternaria alternata, brassicae, brassicicola, daucii, radicina, raphani, 51 solani, tagetica, tenuis, zinniae Bipolaris bicolor, cynodontis, multiformis, nodulosa, oryzae, setaria, 45 sorokiniana, spicifera Curvularia spp. 2 Drechslera avenae, avenacea, bromi, campanulata, dictyoides, 243 erythrospila, graminea, japonica, phlei, tetramera, teres, tritici-repentis, spp. Stemphylium botryosum 1 Ulocladium alternariae, botrytis, consortiale 10 Valsaceae Cryphonectria parasitica 42 Cytospora spp. 9 Diaporthe helianthi, phaseolorum, spp. 93 Phomopsis longicolla, viticola, spp. 27 Ophiostomataceae Leptographium procerum 1 Ophiostoma serpens 8 Botryosphaeriaceae Diplodia sp. 1 Fusicoccum spp. 6 Ceratocystidaceae Ceratocystis fimbriata 1 Thielaviopsis paradoxa 1 Chaetomiaceae Chaetomium globosum, spp. 11 Glomerellaceae Colletotrichum trifolii, spp. 5 Hypocreaceae Gliocladium catenulatum, roseum, spp. 9 Trichoderma aureoviride, hamatum, harzianum, koningii, saturnisporum, 81 viride, spp. Verticillium alboatrum, nigrescens, lecanii, spp. 11 Leptosphaeriaceae Coniothyrium minitans, sp. 3 Phoma betae, sp. 10 Magnaporthaceae Pyricularia oryzae 5 Coniella diplodiella 1 Coryneum cardinale 1 Microascaceae Scopulariopsis sp. 1 Mycosphaerellaceae Aschochyta chrysanthemi, spp. 4 Cladosporium cucumerinum, fulvum, herbarum 3 Septoria apiicola 2 Nectriaceae Cylindrocarpon spp. 5 Fusarium acuminatum, avenaceum, chlamydosporum, compactum, 324 culmorum, equiseti, graminearum, graminum, incarnatum, moniliforme, oxysporum, poae, proliferatum, roseum, sambucinum, semitectum, solani, sporotrichioides, tricinctum, spp. Phaeosphaeriaceae Microsphaeropsis spp. 4 Sclerotiniaceae Botrytis allii, cinerea, gladiolorum, squamosa, tulipae, spp. 22 Monilia spp. 5 Sclerotinia minor, sclerotiorum, spp. 8 Sclerotium cepivorum 2 Trichocomaceae Aspergillus flavipes, fumigatus, ustus, spp. 14 Penicillium expansum, cotylophilum, citrinum, spp. 5 Talaromyces sp. 1 2004 Are terrestrial Ascomycetes lacking in alginolytic activity ? 231

Table 1. (Continued).

Family Genusa Species No. tested isolates

Other Ascomycetes Epicoccum purpurascens 3 Gloeosporium cyclaminis 1 Helminthosporium atypicum 1 Macrophomina sp. 1 Microdochium nivale 10 Stagonospora sp. 1 Ascomycota, Saccaromycetes Dipodascaceae Geotrichum sp. 1 Basidiomycota, Basidiomycetes Bondarzewiaceae Heterobasidion annosum 1 Ceratobasidiaceae Rhizoctonia solani, sp. 6 Oomycota, Oomycetes Pythiaceae Phytophthora cactorum 4

Total 1,107

a Some genera represent the anamorphic stage which teleomorph should be in the concerned Family in Ascomycetes.

More than 1,100 terrestrial fungal isolates, including on such a substrate; in the absence of sucrose many plant pathogenic Ascomycetes or mitosporic As- (medium 3) growth was even more impaired, allowing comycetes (Table 1), maintained at 4°C under mineral the evaluation of the effects of alginate upon colony oil in the culture collection of our department, were morphology according to the growth on medium 2. tested for the ability to grow in the presence of alginate Morphology of colonies developed in presence of al- as sole carbon source, using three different media: (1) ginate as the sole carbon source was never identical 1 Czàpek (Tuite, 1969) containing NaNO3 (2.0 g L ), to that on sucrose, always showing a reduced produc- 1 1 K2HPO4 (1.0 g L ), MgSO4 ·7H2O (0.5 g L ), KCl tion of aerial mycelium and/or sporulation. Most 1 1 (0.5 g L ), FeSO4 ·7H2O (0.01 g L ), sucrose colonies in the presence of alginate showed a mor- (30.0 g L1) and agar (15.0 g L1), as control medium; phology similar to that without any carbon source. (2) Czàpek/alginate, as (1) but without sucrose and Some isolates (27) showed a more compact growth, with 1% soluble sodium alginate (Carlo Erba FU) as and/or a higher production of aerial mycelium, and/or a sole carbon source to test the ability of growing on al- higher sporulation when grown on alginate as the sole ginate and (3) Czàpek/salts as (1) but containing only carbon source than in absence of carbon sources. mineral salts without any carbon source, to determine Production of pigments could be detected, in some the growth of fungal strains in the absence of carbon cases, in the presence of alginate similarly to those sources. All media had pH7.0. The screening was observed in presence of sucrose, whereas no pigmen- performed by plating mycelial disks of 9 mm in diame- tation was detected in absence of carbon sources. All ter cut from the edge of actively growing colonies on these characteristics were considered as signs of de- potato dextrose agar (PDA), at the center of a 10 cm ployment of alginate; thus such isolates were chosen diameter Petri plate filled with each medium. Plates for viscometric testing. The 27 isolates represented were incubated at 24°C for the time needed by 2.5% of the 1,107 isolates tested on agar plates (Table colonies to reach the edge. During incubation the mor- 2). phology of the colonies growing in presence of algi- These isolates belong to three families, Pleospo- nate was observed and compared with that observed raceae, Valsaceae and Ophiostomataceae. Within in the presence of sucrose or in the absence of carbon Pleosporaceae (Pleosporales), Helminthosporium sources. atypicum has been suggested to be similar to species Czàpek (medium 1) is not the best substrate for all within the Bipolaris genus, namely B. multiformis or B. tested fungi as some isolates didn’t show good growth sorokiniana (Alcorn, 1991). One third (4/13) of Bipo- 232 SARROCCO, FANTI, and VANNACCI Vol. 50

Table 2. Species including isolates positive to agar plate test.

Family Genus and species No. tested isolates No. positive isolates

Ascomycota, Ascomycetes Pleosporaceae Bipolaris sorokiniana 12 3 Other Bipolaris spp. 33 — Helminthosporium atypicum 11 Drechslera avenae 17 8 Drechslera teres 39 1 Other Dreschslera spp. 87 — Valsaceae Diaporthe phaseolorum 63 1 Other Diaporthe spp. 30 — Cryphonectria parasitica 42 6 Ophiostomataceae Ophiostoma serpens 86 Leptographium procerum 11

Total isolates 333 27 laris isolates were therefore positive to the agar plate from each species selected by the agar plate test, test (Table 2), but an isolate of B. multiformis (Van- were submitted to viscometric assay. Alginolytic activ- nacci, 1995) tested for alginolytic activity did not give ity was evaluated with a n. 100 Cannon-Fenske vis- positive results. The other genus that gave positive re- cometer. Fungal mycelium (0.2 g) was put in 25 ml of sults was Drechslera. Half of the D. avenae isolates sterile 1% alginate (Carlo Erba FU) water solution, were positive. The two genera, Bipolaris and Drech- supplemented with 0.1% of yeast extract (three repli- slera, are phylogenetically close (Spatafora and Black- cates for each strain). All viscometric measures were well, 1994) performed after 10 days of incubation on a rotatory Only seven Valsaceae () isolates were shaker at 28°C. At the end of the incubation period, positive to the agar plate test and six were Cryphonec- the solution was centrifuged and 7 ml of supernatant tria parasitica, the agent of chestnut canker, but these were pipetted into the mixing chamber of the viscome- last represented only 14.3% of the C. parasitica iso- ter. As a control, Paenibacillus alginolyticus NRRL- lates evaluated. 14423, known for its alginolytic activity (Nakamura, Among Ophiostomataceae (Ophiostomatales), Lep- 1987; Shida et al., 1997) was used. The viscosity val- tographium procerum has been hypothesized to have ues were expressed in centistokes (cSCt; where “C” Ophiostoma as teleomorph (Jacobs and Wingfield, is the calibration constant expressed in centistokes per 2001). Almost all Ophiostoma isolates tested here second, and “t” is the efflux time expressed in sec- were able to grow on alginate as the sole carbon onds). The viscometer used in this work was calibrated source. Some genera were well represented both as and the calibration constant was C0.1401. The Can- number of species and/or as number of isolates non-Fenske viscometer can be used for measuring tested. Among them Fusarium (19 species, 324 iso- viscosity of fluid following a linear kinetic, i.e. for all so- lates; teleomorph should be Gibberella or Nectria) Al- called Newtonian fluid. To obtain this information a 1% ternaria (10 species, 51 isolates), the Diaporthe/Pho- water solution of alginate (Carlo Erba FU) was submit- mopsis complex (4 species, 120 isolates) and Tricho- ted to a kinetic measure using a rheomat (Contraves derma (6 species, 81 isolates) did not show any sign Rheomat 115). Results showed that the solution used of alginate deployment. Trichoderma isolates were for this assay is a Newtonian fluid whose viscosity among those showing poor growth on un-modified could be evaluated by a Cannon-Fenske viscometer. Czàpek. 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