DNA Content of Germinating Spores, Individual Hyphal Cells and Resting Structure Cells of Verticillium spp. Measured by Microdensitometry

Abstract

Using microdensitometry, the cell cycle was investigated in large populations (500) of Feulgen-stained nuclei in conidia, hyphae and resting structures of various strains (all haploid) of Verticillium albo-atrum and V. dahliae as well as a stable diploid strain, V. dahliae var. longisporum. When uninucleate conidia, harvested from 3 d Roux bottle agar cultures, were incubated in stationary liquid complete medium at 24 "C in the dark, they exhibited partial synchrony for germ-tube emergence (50 % emergent at about 7 h, reaching 95 % at 10 to 12 h), DNA replication (50 % increase at about 7.5 h) and mitosis (50 % at about 10 h, reaching 95 % at 12 h); the second nuclear division occurred at 21 to 24 h.Significant amounts of DNA replication were not detected until 6 h ; maximum levels, i.e. a doubling of DNA content (in nuclei not yet divided), occurred at 10 h. There was a 3 h G1 phase between the first and second nuclear divisions ; the S and G2 phases were estimated at about 30 and 90 min duration, respectively. The absolute DNA content per nucleus for ungerminated conidia of one strain of V. dahliae was found to be 0.025 to 0.030pg. In growing hyphae, only the apical cell(s) contained two or more nuclei; DNA levels approximated to those for ungerminated conidia (Gl) but a small number of nuclei had begun the S phase. In hyphal tip regions comprising up to 10 cells, a synchronous nuclear division was observed, and nuclei in the apical cells often had higher DNA values than other nuclei in the same hypha. Cells of young microsclerotia (6 to 8 d-old) of V. dahliae and resting mycelium (9 to 12d-old) of V. albo-atrum were haploid and uninucleate with a small minority of the nuclei in the S phase; no evidence for diploidy or endopolyploidy was found at these sampling levels. g i l h nidulans and Alternaria solani hyphae from radial growth rates and ranges in apical cell length. Journal of General Microbiology 97, CLUTTERBUCK, A. J. (1969). Cell volume per nucleus in haploid and diploid strains of Aspergillus nidulans. Journal of General Microbiology 55, CLUTTERBUCK, A. J. (1970). Synchronous nuclear division and septation in Aspergillus nidulans. Journal of General Microbiology 60, 133-135.

The Ascomycete Verticillium longisporum Is a Hybrid and a Plant Pathogen with an Expanded Host Range

“…A parasexual origin is supported by the observation that nuclei of V. longisporum contain approximately double the DNA of V. dahliae [31], [44]. But there is considerable variation in DNA content between nuclei of different isolates, suggesting differences in chromosome content between isolates.…”

Heterokaryon Incompatibility and Interspecific Hybridization between Verticillium albo-atrum and Verticillium dahliae Following Protoplast Fusion and Microinjection

“…nidulans (Hastie, 1970b;Typas & Heale, 1978), there was at least a 1 000-fold increase of this frequency following protoplast fusion or microinjection ( Table 2). Uninucleate conidia of Verticillium are known to divide almost synchronously under appropriate nutritional conditions (Typas & Heale, 1980), and similar conditions are also known to promote diploid formation in this fungus (Ingle & Hastie, 1974). The high efficiency of diploid formation is therefore probably due to the fact that both methods provide a much more effective system of mixing nuclei, since they allow fusion of many protoplasts or injection of many nuclei into one cell, while most cells in natural heterokaryons are uninucleate.…”

“…Conidia were stained with Feulgen as described by Typas & Heale (1980). Ploidy was inferred from conidial size measurements made with a calibrated eyepiece micrometer; V. dahliae var.…”

Genome Size Determination in Peronosporales (Oomycota) by Feulgen Image Analysis

“…Most data have been obtained by Feulgen absorbance photometry (Typas and Heale, 1980;Jackson and Heale, 1985;Tooley and Therrien, 1987;Therrien et al, 1988Therrien et al, , 1989Therrien et al, , 1993Tooley et al, 1989;Daggett and Goetz, 1992;Daggett et al, 1995); propidium iodide microfluorometry (Eilam et al, 1992) or flow cytometry (Eilam et al, 1994); and DAPI microfluorometry (Whittaker et al, 1991;Rutherford and Ward, 1985). Most of these investigations report only relative DNA values, as their main aim has been to record the relative genome size variation within a species or within closely related taxa, and usually no attempt has been made to report also absolute DNA content values.…”

“…de Bary (2C ϭ 0.52 pg; Tooley and Therrien, 1987), Tilletia indica (Mitra) Mundkur (1C ϭ 0.1 pg; Therrien et al, 1988) and Verticillium dahliae Kleb. (1C ϭ 0.025-0.030 pg; Typas and Heale, 1980). For several fungi, absolute genome sizes have also been estimated by reassociation kinetics (e.g., Achlya bisexualis, with 1C ϭ 0.043 pg; Hudspeth et al, 1977; Aspergillus nidulans, with 1C ϭ 0.028 pg; Timberlake, 1978; Aspergillus flavus and related species, with 1C ranging from 0.034 to 0.040 pg; Kurtzman et al, 1986; the dalton values were converted into picograms using the formula 1 D ϭ 1.66 ϫ 10 Ϫ12 pg according to Cavalier-Smith, 1985: X); also, sometimes genomic reconstruction was applied as a complementary method (e.g., for Bremia lactucae, with 1C of about 0.05 pg; Francis et al, 1990; Phytophthora sojae, with 1C ϭ 0.063 pg; Mao and Tyler, 1991).…”