The Effect of pH on Spore Germination, Growth, and Infection of Strawberry Roots by Fusarium oxysporum f. sp. fragariae, Cause of Fusarium wilt of Strawberry.

Previous work has shown that raising the pH of acidic soil to near neutrality can reduce the incidence of Fusarium wilt. The basis for this effect has not been established. The present study assessed effects of pH on spore germination, growth, and infection of strawberry roots by Fusarium oxysporum f. sp. fragariae, the cause of Fusarium wilt of strawberry. There was not a significant effect of pH (5 versus 7) on the rate of spore germination at either 20 or 25°C for any of the three tested isolates (one representative of each clonal lineage of F. oxysporum f. sp. fragariae found in California). Likewise, pH did not have a significant effect on fungal growth at 20°C. At 25°C, two isolates grew faster at pH 7 than at pH 5. Growth of the third isolate was unaffected by the difference in pH. For the strawberry cultivar Albion, the frequency of root infection was significantly higher for plants grown in acidified soil (near pH 5) than for plants grown in soil near neutrality. The higher frequency of root infection in acidified soil was associated with a lower level of microbial activity, as measured by hydrolysis of fluorescein diacetate.

Fusarium oxysporum f. sp. mori, a New Forma Specialis Causing Fusarium Wilt of Blackberry.

Fusarium oxysporum has recently been identified as the cause of a wilt disease affecting blackberry in California and Mexico. Thirty-six isolates of F. oxysporum obtained from symptomatic blackberry plants in California and Mexico were comprised of nine distinct somatic compatibility groups (SCGs). Phylogenetic analysis of a concatenated data set, consisting of sequences of the translation elongation factor 1-α and ß-tubulin genes and the intergenic spacer of the ribosomal DNA, identified nine three-locus sequence types, each of which corresponded to an SCG. Six SCGs were present only in California, two only in Mexico, and one in both California and Mexico. An isolate associated with the most common SCG in California was tested for pathogenicity on blueberry, raspberry, strawberry, and lettuce. All blueberry, raspberry, and lettuce plants that were inoculated remained healthy, but two of the five strawberry cultivars tested developed symptoms. The three strawberry cultivars that were resistant to the blackberry pathogen were also resistant to F. oxysporum f. sp. fragariae, the cause of Fusarium wilt of strawberry. We propose to designate strains of F. oxysporum that are pathogenic to blackberry as Fusarium oxysporum f. sp. mori forma specialis nov.

The Population of Fusarium oxysporum f. sp. fragariae, Cause of Fusarium Wilt of Strawberry, in California.

The objectives of this study were to investigate the structure of the population of Fusarium oxysporum f. sp. fragariae in California and to evaluate methods for its detection. Fifty-nine isolates of F. oxysporum f. sp. fragariae were obtained from diseased strawberry plants and their identity was confirmed by pathogenicity testing. The full nuclear ribosomal intergenic spacer (IGS) and elongation factor 1-α gene (EF-1α) were amplified by polymerase chain reaction (PCR) and sequenced to elucidate phylogenetic relationships among isolates. IGS and EF-1α sequences revealed three main lineages, which corresponded to three somatic compatibility groups. Primers designed to detect F. oxysporum f. sp. fragariae in Japan amplified a 239-bp product from 55 of 59 California isolates of F. oxysporum f. sp. fragariae and from no nonpathogenic isolates of F. oxysporum. The sequence of this PCR product was identical to the sequence obtained from F. oxysporum f. sp. fragariae isolates in Japan. Intensive sampling at two locations in California showed results of tests based on PCR and somatic compatibility to be in agreement for 97% (257 of 264) of isolates tested. Our findings revealed considerable diversity in the California population of F. oxysporum f. sp. fragariae, and indications that horizontal gene transfer may have occurred.

First Report of Fusarium Stem and Crown Rot of Fennel in Arizona Caused by Fusarium avenaceum.

In 2010 in Yuma, AZ, field-grown fennel (Foeniculum vulgare, Apiaceae) exhibited previously undescribed disease symptoms. The lower stems in contact with soil developed a brown decay and leaves on these stems became chlorotic. White mycelium and orange sporodochia were observed on affected tissues near the soil line. Diseased stems later wilted, died, and resulted in reduced quality of the fennel; these plants were not harvested. Disease distribution was patchy and prevalence was approximately 5%. Symptomatic tissues were surface sterilized in a dilute (1%) bleach solution for 3 min and tissues from the margins of the decay were placed into petri plates containing acidified corn meal agar (2 ml of 25% lactic acid/liter). Isolations consistently resulted in the recovery of a presumptive Fusarium species. Isolates were transferred to carnation leaf agar and incubated at 22°C under fluorescent lights for 10 days. Morphologies of all isolates were identical, with macroconidia being long and slender, slightly curved, with elongated, bent apical cells and notched basal cells. Conidia were borne on monophialides. Microconidia were sparse and chlamydospores were not observed. For two isolates, a portion of the translation elongation factor 1-alpha gene (TEF) was amplified with primers ef1 and ef2 (3). Based on a comparison of 668 base pairs, both isolates had the same sequence, which differed by one base pair from an accession (GQ915502.1) of Fusarium avenaceum in GenBank. The same single base pair also separated the two fennel isolates from an isolate of F. avenaceum (GL 13) previously recovered from Eustoma grandiflorum (=Lisianthus russellianus) (2). Thus, both morphological and molecular criteria support identification of the recovered fungus as F. avenaceum (Fries) Saccardo. Partial TEF sequences were deposited in GenBank (Accession Nos. JN254784, JN254785, and JN254786 for the two fennel isolates and GL 13, respectively). All isolates are archived in the Department of Plant Pathology at University of California, Davis. Pathogenicity was tested by cutting shallow slits into fennel stems, inserting one colonized agar plug into each cut, and wrapping the stems with Parafilm. Five isolates from fennel were tested on 10 stems each. Control plants were inoculated with uncolonized agar plugs. Plants were maintained at 24 to 26°C in a greenhouse. After 6 to 8 days, a brown decay developed on 70 to 90% of Fusarium-inoculated stems at the points of inoculation. Foliage later became chlorotic and F. avenaceum was recovered from all symptomatic stems. Control plants were symptomless. The experiment was completed two times and results were the same. In addition, F. avenaceum isolate GL13 from E. grandiflorum (2) was inoculated onto fennel plants with the same method. However, these inoculated plants remained symptomless. To our knowledge, this is the first report of a stem and crown rot disease of fennel caused by F. avenaceum. Apparently, the only other published account of a Fusarium disease of fennel is root rot caused by F. solani (1). The inability of the Eustoma isolate of F. avenaceum to cause disease in fennel suggests that these two crown rot pathogens may have restricted host ranges. References: (1) J. H. Gupta and V. P. Srivastava. Indian J. Mycol. Plant Pathol. 8:206, 1979. (2) S. T. Koike et al. Plant Dis. 80:1429, 1996. (3) K. O'Donnell et al. Proc. Nat. Acad. Sci. U.S.A. 95:2044, 1998.

Fusarium Wilt of Strawberry Caused by Fusarium oxysporum in California.

Beginning in 2006 and continuing into 2009, an apparently new disease of strawberry (Fragaria × ananassa) affected commercial plantings (cvs. Albion, Camarosa, and others) in coastal (Ventura and Santa Barbara counties) California. Symptoms consisted of wilting of foliage, drying and withering of older leaves, stunting of plants, and reduced fruit production. Plants eventually collapsed and died. Internal vascular and cortical tissues of plant crowns showed a brown-to-orange-brown discoloration. Differences in cultivar susceptibility were not recorded. Internal crown and petiole tissues, when placed on acidified corn meal agar, consistently yielded Fusarium isolates having similar colony morphologies. No other pathogens were isolated. The Fusarium isolates were subcultured on carnation leaf agar and observed to be producing macroconidia and microconidiophores that are diagnostic of Fusarium oxysporum (1). For two of these isolates, the internal transcribed spacer region comprising ITS1, ITS2, and 5.8S rRNA was amplified using primers ITS-1 and ITS-4 (3). On the basis of a comparison of 515 bp, both isolates had the identical sequence, which was a 100% match for 30 accessions of F. oxysporum in GenBank. This comparison included several formae speciales of F. oxysporum, but F. oxysporum f. sp. fragariae, a previously described pathogen of strawberry (4), was not included. The isolates are archived in the Department of Plant Pathology at UC Davis and are available on request. Both sequenced isolates plus four others were tested for pathogenicity on strawberries. For these tests, spore suspensions of 1 × 105 conidia/ml were prepared separately for six isolates. Roots of strawberry transplants (12 plants of cv. Camino Real) were cut and soaked in spore suspensions for 10 min. Plants were potted in soilless, peat moss-based medium in containers. Control strawberry plants were soaked in water prior to planting. All plants were then grown in a shadehouse. After 8 weeks, inoculated plants began to show wilting and decline of foliage and internal crown tissue was lightly discolored. F. oxysporum was isolated from all inoculated plants. Control plants did not exhibit any disease symptoms and crown tissue was symptomless. To our knowledge, this is the first report of Fusarium wilt of strawberry in California. This disease has been reported from a number of other countries including Argentina, Australia, China, South Korea, Spain, and Japan (2). Since 2006, Fusarium wilt of strawberry has increased in incidence and severity in California. Initial problems in 2006 consisted of multiple small patches (2 to 4 beds wide × 3 to 10 m long) of diseased plants; in these patches disease incidence could range from 80 to 100%. By 2009, in some fields, the disease affected large sections that ran the length of the field. References: (1) P. E. Nelson et al. Fusarium Species: An Illustrated Manual for Identification. Pennsylvania State University Press, University Park, 1983. (2) H. S. Okamoto et al. Plant Prot. 24:231, 1970. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Application. Academic Press, NY, 1993. (4) B. L. Winks and Y. N. Williams. Qld. J. Agric. Anim. Sci. 22:475, 1966.

Limiting Effects of Low Temperature on Growth and Spore Germination in Gibberella circinata, the Cause of Pitch Canker in Pine Species.

Pitch canker, caused by Gibberella circinata (anamorph = Fusarium circinatum), causes canopy dieback and mortality in susceptible pine species in many parts of the world. Pitch canker is most problematic in areas with a relatively warm climate, suggesting a possible limitation on disease development imposed by low temperatures. To test this hypothesis, the effect of temperature on radial growth was examined in isolates of G. circinata of diverse geographic origin. All isolates grew most rapidly at 25°C and progressively more slowly at 20, 15, and 10°C. Spore germination occurred most rapidly at 20°C and was slowest at 10°C. To determine if the time required for spore germination might influence the likelihood of infection, the duration of wound susceptibility was examined by inoculating branches of susceptible Monterey pines (Pinus radiata). In each of six field trials, branches were wounded and then inoculated immediately or at 2, 6, or 9 days after wounding. The results indicated that wounds inoculated immediately became infected at a significantly higher rate than those inoculated 2 days later. Thus, if low temperatures extend the time required for germination beyond this period, a reduced infection frequency would be expected. Such a limiting effect of temperature could help to explain the current distribution of pitch canker.

First Report of the Pitch Canker Fungus (Fusarium circinatum) in the Sierra Nevada of California.

The pitch canker fungus, Fusarium circinatum (teleomorph Gibberella circinata), was isolated from a branch originating from rootstock of a Douglas-fir (Pseudotsuga menziesii) graft in a breeding orchard at 1,000m elevation in El Dorado County, California. We visited the orchard after the New Zealand Ministry of Agriculture and Forestry reported in November 2003 that a Douglas-fir scion (branch cutting) shipped from there in January-and subsequently grafted and held in a quarantine facility near Christchurch-was infected with the pitch canker fungus. We took samples throughout the orchard of any branches that appeared unhealthy. In addition, asymptomatic branches from the tree alleged to be the source of the New Zealand infestation were collected to assay for propagules of F. circinatum. Wash water from these branches was negative for the pathogen. Likewise, F. circinatum was not recovered from water washings of 20 branches collected randomly throughout the orchard. Fifteen branch samples collected from symptomatic Douglas-fir grafts were cultured on water agar and only one yielded a colony with an appearance consistent with F. circinatum. A single spore subculture of this isolate was confirmed as F. circinatum on the basis of colony morphology and the structure of the microconidiophores (1). The virulence of this isolate was evaluated by inoculating susceptible 2-year-old Monterey pine (Pinus radiata) seedlings with a toothpick to wound the main stem and insert potato dextrose agar colonized by the fungus. Twenty-four days later, pitching and yellow needles were evident at the site of inoculation, and removal of the bark revealed resin-soaked and discolored tissue. Concurrent with the pathogenicity test described above, a culture of the putative F. circinatum isolated in New Zealand was inoculated into Monterey pines with an identical outcome. The fungus was reisolated from lesions from both sets of inoculations and confirmed as F. circinatum based on morphological criteria. Isolates GL285 and GL286 are available from T. R. Gordon upon request. Prior to its discovery in the Sierra Nevada, pitch canker in California was known only from counties on or near the coast. Our report indicates the pathogen can survive and infect trees 110 km east of the previous most-inland site of infestation. It remains to be seen how extensively pitch canker will develop in the Sierra Nevada. Douglas-fir is only moderately susceptible to F. circinatum, which has not been observed to cause significant damage to this species. On the other hand, low-elevation Sierra Nevada pines including P. sabiniana, P. coulteri, and P. ponderosa are substantially more susceptible than are Douglas-fir in greenhouse tests (2). References: (1) T. R. Gordon et al. Mycol. Res. 100:850, 1996. (2) T. R. Gordon et al. Plant Dis. 85:1128, 2001.