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Tropical Plant Pathology

On-line version ISSN 1983-2052

Trop. plant pathol. vol.39 no.3 Brasília May/June 2014 



First report and characterization of Fusarium circinatum, the causal agent of pitch canker in Brazil



Ludwig H. PfenningI; Sarah da Silva CostaI; Maruzanete Pereira de MeloI; Hélcio CostaII; José Aires VenturaII; Celso Garcia AuerIII; Álvaro Figueredo dos SantosIII

IDepartamento de Fitopatologia, Universidade Federal de Lavras, Cx. Postal 3037, 37200-000, Lavras, MG, Brazil
IIInstituto Capixaba de Pesquisa, Assistência Técnica e Extensão Rural -INCAPER, 29052-010, Vitória, ES, Brazil
IIIEMBRAPA Florestas, 83411-000, Colombo, PR, Brazil

Author for correspondence




Pitch canker is one of the most important diseases of pine trees worldwide, including South America. The causal agent of this disease is Fusarium circinatum, a member of the Gibberella fujikuroi complex (GFC). In South America, the species is reported from Colombia, Uruguay and Chile, but is considered a quarantine organism in Brazil. In this study we characterized isolates obtained from symptomatic pine seedlings intercepted in a nursery in Santa Catarina State using phylogenetic analyses, crossings and morphological markers. The Brazilian isolates grouped with reference material in an unique clade and formed fertile perithecia when crossed with reference tester strains. The detailed characterization given here and the availability of tester strains will allow for reliable identification and support monitoring of this important plant pathogen.

Key words: Pinus, Gibberella fujikuroi species complex, mating population, molecular phylogeny.




International trade has long been recognized as a major conduit by which nonindigenous plant pests arrive in Brazil and other countries. Invasive plant pathogens have dramatically affected the crops productivity and function of natural and agricultural ecosystems. Pitch canker of pine (Pinus spp.) is caused by Fusarium circinatum Nirenberg & O'Donnell and is frequently associated with high levels of mortality in nurseries and on mature trees, resulting in significant economic losses (Wingfield et al., 2008). The disease was first discovered in the southeastern United States (Hepting & Roth, 1946; Gordon et al., 2001) and the causal agent described as F. subglutinans f. sp. pini (Correll et al., 1991). The pathogen was then recorded in numerous countries worldwide, including Mexico, Colombia, Chile and Uruguay (Britz et al., 2001; Wingfield et al., 2002; Alonso & Bettucci, 2009; Steenkamp et al., 2012). F. circinatum is known to infect more than 50 species of pines (Wingfield et al., 2008). Symptoms of infection in nurseries include damping-off and wilting of seedlings. On mature trees, pitch canker is characterized by heavy exudation of resin at the site of infection (Wingfield et al., 2008) although other plant parts such as roots, shoots, female flowers and mature cones, seed and seedlings can also be affected (Viljoen et al., 1994).

This fungus is a member of the Gibberella fujikuroi complex (GFC) and is known to reproduce both sexually and asexually. The sexual form Gibberella circinata is cross-fertile under laboratory conditions and represents a distinct mating population (MP-H) in the GFC (Britz et al., 1998; 1999). Morphological characters like the production of sterile coiled hyphae are not consistent and therefore reliable identification must be complemented by a phylogenetic analysis or laboratory crosses (Leslie & Summerell, 2006). Here we report the occurrence of Fusarium circinatum during an interception in Brazil and characterize local strains by means of phylogeny, crossings, morphological characters and pathogenicity tests.



Cultures and culture conditions

The present study included strains isolated from diseased Pinus taeda seedlings collected in a nursery in Santa Catarina State, Brazil and reference strains of F. circinatum (MRC 7488 -MAT1-1; MRC 6213 -MAT1-2; NRRL 25331 - Type strain). Pinus taeda seedlings produced from imported seeds showed symptoms of wilting and some were killed by the disease, and isolates obtained from symptomatic plants were initially identified as Fusarium subglutinans (Grigoletti Júnior et al., 2006). All evaluated strains were used as single spore cultures and are deposited as spore suspension in 15% glycerol at -70ºC at CML -Coleção Micológica de Lavras, Universidade Federal de Lavras (Table 1).

Morphological and cultural studies

Potato dextrose agar (PDA) was used to evaluate characters of the colony and calculate the growth rate. Morphological characteristics of the strains were observed on synthetic nutrient-poor agar (SNA). Cultures were incubated for 10 to 14 days at 20ºC in a 12h photoperiod under near UV and white fluorescent bulbs. The strains were cultivated at 20ºC on PDA in the dark for determination of growth rates and color. Colony diameters were measured on three plates of each culture after 4 days of incubation (Nirenberg & O'Donnell, 1998; Leslie & Summerell 2006).

Sexual compatibility and laboratory crosses

A MAT1-1 allele fragment was amplified using the primer set Gfmat1a and Gfmat1b, and a MAT1-2 allele fragment was amplified with the Gfmat2c and Gfmat2d primers (Steenkamp et al., 2000). Strains of opposite mating types were intercrossed and also with tester type strains of Fusarium circinatum (MRC 7488 -MAT1-1; MRC 6213 - MAT1-2) (Leslie & Summerell, 2006). Strains were grown in tubes with complete medium for one week at 20ºC under 12-h photoperiod to be used as male parents. Conidial suspensions were prepared by adding 2 mL sterile distilled water. Tester strains used as female parents were grown on carrot agar (CA) plates for one week at 25ºC in the dark and fertilized by adding 2 mL of the conidial suspensions of the Brazilian strains onto the surface of the CA plates, spreading and mixing vigorously all over the surface (Klittich & Leslie, 1988; Leslie & Summerell, 2006). The plates were incubated upright at 20ºC under 12-h photoperiod. All crosses were repeated twice. Crosses were evaluated weekly during six weeks for formation of perithecia and ascospore exudation. A cirrus of ascospores was collected from three perithecia of each fertile cross, suspended in sterile water, and spread on the surface of 2% water agar in a Petri dish. Plates were incubated overnight at 25ºC in the dark and checked for ascospore germination (Lima et al., 2012). Special care was taken to avoid transfer of conidia with ascospores.

DNA extraction, PCR amplification and sequencing

Monospore cultures were grown in complete medium broth (CM; Correll et al., 1987), harvested by filtration, and DNA was extracted with a standard CTAB protocol (Leslie & Summerell, 2006). Portions of the cmd gene were amplified from representatives of F. circinatum and F. sterilihyphosum with primers CL1 (forward; 5'-GAR TWC AAG GAG GCC TTC TC-3') and CL2A (reverse; 5'-TTT TTG CAT CAT GAG TTG GAC-3') using the amplification conditions described by O'Donnell et al. (2000). Portions of the tef1 gene were amplified by using primers Ef-1 (forward; 5'-ATG GGT AAG GAG GAC AAG AC-3') and Ef-2 (reverse; 5'-GGA AGT ACC AGT GAT CAT GTT-3') with the amplification conditions of O'Donnell et al. (1998). PCR products were stained using Gel-Red stain (Biotium), separated by electrophoresis in 1% agarose gels, and visualized under UV light (312 nm). PCR amplified fragments were purified with a Gen Elute PCR cleanup kit (Sigma-Aldrich) and sequenced in both directions on an automated MEGA BACE 1000 capillary sequencer (GE Healthcare).

Sequence alignment and phylogenetic analyses

Consensus sequences were assembled from forward and reverse sequences using SeqAssem ver. 07/2008 (SequentiX -Digital DNA Processing). Additional sequences of other species of the Gibberella fujikuroi complex were obtained from Genbank (Altschul et al., 1997). Sequences were aligned using ClustalW as implemented by Mega 5 (Tamura et al., 2011). Maximum Parsimony (MP) phylogenetic analysis was performed on Mega 5 (Tamura et al., 2011) using the Close-Neighbor-Interchange algorithm with search level 1, and ten initial trees were obtained with the random addition of sequences. Clade support was inferred from 1000 bootstrap replications. All positions containing gaps were eliminated from the analysis (Phoulivong et al., 2010). Sequences of F. oxysporum NRRL 22902 and F. inflexum NRRL 20433 were used to root the trees based on prior analyses of the GF-C.

Pathogenicity tests

The pathogenicity tests were conducted with two of the Brazilian isolates (CML 2128, CML 2369), and one strain of Fusarium tupiense (CML262) as control. Seedlings of Pinus patula were grown in the greenhouse at 26±3ºC under natural conditions, and then transplanted to 5 L pots containing a 50:50 mixture of soil and a commercially available substrate (Plantmax). Inoculum was prepared by culturing each isolate on 6-cm plastic Petri dishes containing Malt Extract 2% for 1 week at 25ºC in the dark, after which agar plugs 6 mm in diameter were excised from the growing edge of the colony for inoculation. Wounds were made in the upper third of the stem using a fine steel needle. The plugs with mycelium were placed on the wounds and covered with plastic film to prevent desiccation (Roux et al., 2007). Pots were then placed in the greenhouse under natural photoperiod at 26±3ºC for 5 weeks. Treatments were arranged in a completely randomized design with five replications. Not inoculated plants served as a negative control. Assessments were made weekly, during 8 weeks. Inoculation experiments were conducted twice in time.



Strains obtained from Pinus seedlings in Santa Catarina were identified as Fusarium circinatum based on phylogeny, laboratory crosses and morphological features. Amplified DNAsequences of the tef1andcmdgene fragments were aligned with the sequences of reference strains and other species of the GFC available from GenBank. In all tree topologies generated by Maximum Parsimony (MP), in separate analyses of cmd and tef1 as well as in the combined analysis, isolates from Pinus in Brazil clustered together with the type strain of F. circinatum in a strongly supported clade (99%) (Figure 1).

The presence of both mating idiomorphs from the Brazilian isolates was confirmed by amplification of the MAT loci. Two isolates belong to MAT1-1 mating type idiomorph (CML 2128, CML 2369), whereas two others contained the MAT1-2 mating type idiomorph (CML 2367, CML 2368). Perithecia were observed after four weeks in all crosses between the individual tester strains of MP-H (MRC 7488, MAT1-1; MRC 6213, MAT1-2) and the strains of Fusarium associated with Pinus in Brazil (Figure 2E). The morphological characteristics of perithecia, ascus and ascospore were consistent with those described in the literature (Nirenberg & O'Donnell, 1998; Britz et al., 2002). About 80-90% of ascospores germinated within 24 h of transfer to water agar.

Cultures showed sterile hyphae in the aerial mycelium, monophialides and polyphialides with two or more openings on proliferating conidiophores, and produced obovoid conidia aggregated in false heads in the aerial mycelium, mostly non-septate or occasionally with one septum. Macroconidia were produced in sporodochia, pale orange in color, relatively slender with no significant curvature and usually tri-septate (Figure 2A-D). No chlamydospores were observed. Colonies grown on PDA had a lanose aspect, with white or slightly pink aerial mycelium and appearing salmon colored from the underside of the plate, eventually with a violet pigment in the agar. The average mycelial growth rate was 5 mm/d at 20ºC.

In the pathogenicity tests, isolates induced typical symptoms after about 21 days of inoculation. The infected seedlings showed discolored lesions with yellowing of needles evolving to burning and leaf fall. After 35 days, the seedlings showed symptoms of dieback in the upper part (Figure 2F). The isolate of F. tupiense didn't induce symptoms on inoculated plants. To fulfill Koch's postulates, the pathogen was re-isolated from inoculated plants and identified as F. circinatum based on morphology.



Results of phylogenetic analyses, mating compatibility tests, evaluation of morphological markers and pathogenicity tests showed that the fungus associated with diseased seedlings of Pinus in Brazil is F. circinatum. The application of different species concepts allowed for a reliable and consistent identification of this species, which is a member of the GFC, where morphological markers are scarce or sometimes even absent (Kvas et al., 2009; Lima et al., 2012). The species was first described as a special form of F. subglutinans, as it shares the main morphological features with this species, like the presence of polyphialides, production of conidia in the aerial mycelium only in false heads and the absence of chlamydospores (Correll et al., 1991; Nirenberg & O'Donnell et al., 1998). Morphological characteristics typical of F. circinatum like conidiophores on the erect aerial mycelium, polyphialides with more than two conidiogenous openings and sterile coils could be observed, but these characters are shared with species like F. mexicanum, F. pseudocircinatum, F. sterilihyphosum and F. tupiense (Nirenberg & O'Donnell, 1998; Britz et al., 2002; Lima et al., 2012).

All sexual crosses of strains from Brazil with mating tester strains of F. circinatum produced the sexual stage. These crosses between the individual tester strains of the MP-H from South Africa and strains from Brazil evidence that no reproductive isolation between both populations exists. Moreover, the presence of both MAT idiomorphs indicates that sexual reproduction may occur in Brazil. The sexual reproduction increases the genetic variability of the pathogen causing potential consequences for breeding programs and the management of this pathogen.

Pathogenicity tests revealed that strains recovered from symptomatic Pinus patula in Brazil were pathogenic to P. patula. The pathogen survives in soil and can be seedborne, so there can be extensive mortality of seedlings in planted and natural Pinus stands. Also, pitch canker is frequently associated with a reduction in growth volume and is often associated with significant economic losses (Viljoen et al., 1994; Storer et al., 1998; Wingfield et al., 2008). Fusarium circinatum was recently reported in association with grass species as symptomless hosts in the proximity of Pinus stands (Sweet & Gordon, 2012). The ability to survive in alternative hosts is relevant for establishment and subsequent dissemination of the pathogen to new areas. The movement of infested seeds could also explain the introduction of F. circinatum to new areas (Gordon, 2006).

In South America, pitch canker and its causal agent were reported previously in Chile, Uruguay and Colombia (Wingfield et al., 2002; Alonso & Bettucci, 2009; Steenkamp et al., 2012). We suggest that the pathogen was introduced to the nursery in Brazil on seeds from external sources. Rouging (eradication) of diseased material in nurseries may have prevented the spread of this fungus. No plants with symptoms of pitch canker were found in subsequent surveys for diseased plants in nurseries and plantations of Pinus. Nevertheless, introduction of this pathogen to new areas in recent years calls for strict monitoring and sanitation practices (OEPP/EPPO, 2005).

This study represents the first confirmed report F. circinatum in Brazil, which is considered a quarantine fungus and one of the most serious pathogens of Pinus. This complete characterization of the pathogen and available tester strains for laboratory crosses will allow for reliable identification and support future monitoring of this pathogen in Brazil. New surveys should be conducted in areas planted with Pinus in southern Brazil because the presence of the pathogen is a major threat to the commercial use of pines.



The authors are grateful to John F. Leslie and Hester F. Vismer for providing reference materials and valuable information. This research received partial support from Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq (Proc. 484307/2010-0) and Fundação de Amparo à Pesquisa do Espírito Santo -FAPES. LHP acknowledges the fellowship given by CNPq, SSC a grant provided by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES-PNPD.



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Author for correspondence:
Ailton Reis
Ludwig H. Pfenning

Submitted: 14 October 2013
Revisions requested: 19 November 2013
Accepted: 22 December 2013



Section Editor: Thomas Harrington

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