CHAPTER 2 CHARACTERIZATION OF THE PITCH CANKER FUNGUS, FUSARIUM CIRCINATUM FROM MEXICO
Fusarium circinatum (=F. subglutinans f. sp. pini) is the causal agent of pitch canker of pines. This fungus occurs in the United States, Japan, Mexico and South Africa and it can be introduced into new areas on seed and infected plant material. Its presence in cones from symptomless trees is of concern, particularly with respect to seed transmission. In this study, isolates of Fusarium spp. were collected from Pinus patula, P. greggii, P. teocote and P. leiophylla trees in Mexico, showing typical symptoms of pitch canker, as well as from cones from apparently healthy trees. Morphological characteristics of the pitch canker fungus and isolates of F. subglutinans from other hosts are very similar. Therefore, pathogenicity tests, sexual compatibility studies and histone H3-RFLPs were used to characterize isolates. Isolates collected from Pinus spp. from Mexico were identified as F. circinatum. In this study we have thus confirmed that F. circinatum occurs on pines in Mexico and that the affected trees can be asymptomatic.
Pitch canker, caused by Fusarium circinatum Nirenberg & O’Donnell (=F. subglutinans(Wollenweber & Reinking) Nelson et al. f. sp. pini Correll et al.), was first reported in the southeastern United States (Hepting & Roth, 1946). F. circinatum is now found throughout this region where it has caused significant losses on a wide variety of pine species. This led to the suggestion that pitch canker is endemic to the area (Dwinell et aI., 1985). More recently, pitch canker was identified and reported in California (McCain et aI., 1987), predominately on Pinus radiata planted in landscape settings (Correll et al., 1991). Since 1992, it has been recognized as a threat to native P. radiata stands in California (Storer et aI., 1994; Storer et al., 1998). F. circinatum is also found in Japan (Muramoto et al., 1988; Kobayashi & Kawabe, 1992) and South Africa (Viljoen et al., 1994; Viljoen et aI., 1997).
In South Africa, the fungus was reported from forestry nurseries where it has resulted in serious losses of P. patula (Viljoen et al., 1994), P. elliottii, P. greggii and P. radiata seedlings. Stern cankers on larger trees such as those found in the United States (Hepting & Roth, 1946; Dwinell et al., 1977) have not been seen in South Africa (Wingfield et al., 1999). Pitch canker has been reported in Mexico on a variety of native pine species (Santos & Tovar, 1991; Guerra-Santos, 1999) and this is thought to be the origin of the pathogen, for areas such as South Africa where Mexican pines are commonly propagated. Fusarium circinatum has been isolated from stems and branches of trees (Hepting & Roth, 1946; Dwinell et al., 1977), root collars of pine seedlings (Barnard & Blakeslee, 1980; Viljoen et al., 1994), female strobili, mature cones and seeds (Dwinell et al., 1977; Miller & Bramlett, 1978; Dwinell et al., 1985; Barrows-Broaddus, 1986; 1987; Storer et al., 1998; Dwinell, 1999). Recently, Storer et al. (1998) isolated F. circinatum from pine seedlings originating from seeds collected from cones on diseased as well as asymptomatic branches. Those authors hypothesized that F. circinatum can be carried inside seeds and may remain dormant until germination, which increases the possibility of seedling infections. The implication of seed transmission is serious, since current treatments may be ineffective in eliminating the pathogen. This would increase the possibility of introducing the pathogen into uninfested areas (Barrows-Broaddus & Dwinell, 1985; Storer et al., 1998). Fusarium subglutinans sensu lato includes species occurring on a wide variety of hosts, including pineapple, maize, mango, pine and sugarcane (Booth, 1971). Correll et al. (1991) distinguished pine and non-pine F. subglutinans isolates based on pathogenicity to pines. Those authors proposed that F. subglutinans from pine should be designated as F. subglutinans f. sp. pini based on its exclusive pathogenicity to pine trees (Correll et al., 1991). Restriction fragment patterns of the mtDNA and random amplified polymorphic DNA (RAPD) also indicated that pine isolates differed from non-pine isolates (Correll et a!., 1992; Viljoen et al., 1997). Sexual compatibility among isolates causing pitch canker on pines confirmed that this group corresponded to a distinct biological species (Viljoen et al., 1997). O’Donnell et al. (1998) showed pine isolates to be phylogenetic ally distinct and Nirenberg & O’Donnell (1998) thus proposed the name, F. circinatum, for it. Steenkamp et al. (1999) could, furthermore, distinguish F. circinatum from other species in F. subglutinans sensu lato using histone H3 gene sequences.
Until recently, the most reliable technique to distinguish F. circinatum from closely related Fusarium spp. has been sexual compatibility. A molecular technique based on RFLP profiles of the histone H3 gene, reliably and rapidly distinguishes F. circinatum from other similar Fusarium spp. in the Gibberella jUjikuroi (Sawada) Ito in Ito & K. Kimura complex (Steenkamp et al., 1999). Sexual compatibility as well as histone H3-RFLPs can, therefore, be used to separate the eight different mating populations (biological species), designated by the letters A to H, in this complex. Heterothallic F. circinatum isolates reside in mating population H of the G. jUjikuroi complex and
tester strains representing opposite mating types have been selected and designated (Coutinho et al. 1995; Britz et al., 1998; 1999). Sexual compatibility of field isolates with tester strains of mating population H (MRC 6213 and MRC 7488) provides a firm basis for the identification of field isolates as F. circinatum. In this study, isolations from pine trees in Mexico showing typical canker symptoms
were made. The possible association of F. circinatum with asymptomatic cones was also investigated. The identity of these isolates was verified using morphology (Nelson et al., 1983; Nirenberg & O’Donnell, 1998) as well as pathogenicity, sexual compatibility and histone H3-RFLP comparisons.
MATERIALS ET METHODS
Isolates, cultural and morphological characteristics Fusarium circinatum strains (MRC 7568-7587) were collected in Laguna Atezca and Hidalgo, Mexico, from cankers occurring on branches of native stands of P. patula and P. greggii (Table I). Strains MRC 7568-7569 were isolated from apparently healthy cones collected from asymptomatic P. patula trees and strains MRC 7570- 7587 were obtained from branches showing pitch canker symptoms. Strains MRC 7570-7572, MRC 7573-7576, 7577-7579, 7580-7582 and 7583-7585 were isolated from five trees. Strains MRC 7588-7601 were isolated from cankers on branches of P. teocote and P. leiophylla in northern Michoacan, Mexico (Table 1). Single conidial isolates of all cultures were prepared and are maintained in 15% glycerol at -70°C in the Fusarium collection of the Tree Pathology Co-operative Programme (TPCP), Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa. Isolates have also been deposited in the culture collection of the Medical Research Council (MRC), P. O. Box 19070, Tygerberg, South Africa.
For isolations, pine cones were immersed in 70% ethanol for 2 min. Small pieces (approximately 5 mm2 ) of the asymptomatic cone scale tissue and wood pieces (2 mm2 ) from the infected tree branches were removed and plated on 2% malt extract agar (MEA). Fungi were allowed to grow for seven days at room temperature. Small agar pieces (approximately 5 mm2 ) from the edges of the colonies were transferred to 90 mm plastic Petri dishes containing carnation leaf agar (CLA) (Fisher et al., 1982).
Cultures were incubated at 23°C under near-ultraviolet and cool-white light with a 12 h photoperiod to stimulate culture and conidium development. Isolates were identified and morphological features were compared with F. circinatum tester strains (MRC 6213 and MRC 7488) after 10 to 14 days, using a light microscope. Diagnostic characteristics used were those specified by Nirenberg & O’Donnell (1998) and Nelson et al. (1983).
All isolates obtained from Mexico (Table 1), as well as, F. circinatum tester strains (MRC 6213 and MRC 7488) from South Africa were used in sexual compatibility tests and for comparison based on histone H3-RFLPs. F. sacchari (Butler) W. Gams (= F. subglutinans) and F. subglutinans standard tester strains of the B (MRC 6524 and MRC 6525) and E (MRC 6483 and MRC 6512) mating populations of the G. jujikuroi complex were also included. Tester strains of the B mating population are progeny from a fertile cross between two isolates from sugarcane (Saccharum officinarum) from Hsingying, Taiwan and tester strains of the E mating population were isolated from maize (Zea mays) in St. Elmo, Illinois, USA (Leslie, 1991). F. subglutinans isolates from maize (MRC 1077) and mango (Mangifera indica) (MRC 7035) were included in the histone H3-RFLP comparisons as representatives of host specific isolates of F. subglutinans (Table 1).
RFLPs of Histone H3 gene
DNA was extracted from cultures (Table 1) grown for 10 days in complete medium broth (CM) (Correll et al., 1987) using the protocol described by Steenkamp et al. (1999). PCR reactions were performed using primers (H3-1a and H3-1b) for amplification of the Histone H3 gene PCR product as described by Glass & Donaldson (1995). The same conditions as those described by Steenkamp et al. (1999) were used except Boehringer Mannheim polymerase and reaction buffers
(Boehringer Mannheim South Africa Pty. Ltd.) were used. The restriction enzyme, Dde I, which distinguishes F. circinatum isolates from other similar Fusarium spp. (Steenkamp et al. 1999), was used in this study. Digests were performed using this restriction enzyme in a total reaction volume of 20 III containing 5 U of the enzyme. Sodium chloride was added to the reactions with the enzyme Dde I to a final concentration of 100 mM. All digestion reactions were incubated at 37°C for 7 h. Restriction fragments were separated using agarose gels (Promega Corporation, Madison, Wisconsin, USA) in the presence of ethidium bromide (0.1 Ilg/ml). RFLP fragments were electrophoresed on 3% (w/v) agarose gels and visualized using an ultraviolet transilluminator (Ultra-Violet Product). The visualized RFLP fragments were photographed using a gel documentation system (Microsoft Corporation) and evaluated using the methods described by Steenkamp et al. (1999).