Seed Transmission of Fusarium verticillioides in Maize Plants Grown Under Three Different Temperature Regimes.

Fusarium verticillioides can be seed transmitted and cause systemic infection of maize; however, the frequency of these phenomena has varied widely among and within individual studies. In order to better understand this variability, we evaluated the effect of temperature on the first step in the systemic infection process, the transmission of F. verticillioides from seed to seedling. Seed of a commercial maize hybrid were inoculated with a strain of F. verticillioides that had been transformed with a gene for green fluorescent protein (GFP). The seed were planted in a greenhouse potting mix and incubated in growth chambers. Plants were incubated at one of three temperature regimes designed to simulate average and extreme temperatures occurring in Iowa during the weeks following planting. Root, mesocotyl, and stem tissues were sampled at growth stages V2 and V6, surface disinfested, and cultured on a semiselective medium. At V2, >90% of root and mesocotyl tissues was infected by the GFP-expressing strain at all three temperature regimes. Also at V2, infection was detected in 68 to 75% of stems. At V6, infection of root and mesocotyl tissues persisted and was detected in 97 to 100% of plants at all three temperature regimes. Plants also had symptomless systemic infection of belowground and aboveground internodes at V6. Infection of the three basal aboveground internodes was 24, 6, and 3% for the low-temperature regime; 35, 9, and 0% for the average-temperature regime; and 46, 24, and 9% for the high-temperature regime. Seed transmission and systemic infection occurred at all temperatures and did not differ significantly among treatments. These results indicate that, if maize seed is infected with F. verticillioides, seed transmission is common and symptomless systemic infection can be initiated under a broad range of temperature conditions.

Genotypes A and B of Cadophora gregata Differ in Ability to Colonize Susceptible Soybean.

Growth chamber experiments were conducted to determine if extent of colonization of soybean stems by genotypes A and B of Cadophora gregata (Phialophora gregata), the causal agent of brown stem rot (BSR) of soybean, is similar in soybean plants resistant or susceptible to genotype A. Upon introduction of the two genotypes separately into the base of stems of 2-week-old seedlings, genotype A advanced with the growing tips of susceptible but not resistant genotypes. In contrast, genotype B did not advance with the growing tips of either resistant or susceptible soybean. In similar experiments, 5 weeks after introduction of genotype A, both mean percent stem length colonized by C. gregata and mean percentage of symptomatic trifoliate leaflets were significantly less for resistant than for susceptible genotypes. For genotype B, there was no or a slight difference between resistant and susceptible soybean genotypes in mean percent stem length colonized and no difference in mean percentage of symptomatic trifoliate leaflets 5 weeks after introduction of the pathogen. These results indicate that genotype A and genotype B differ not only in the severity of foliar symptoms they cause on genotype A-susceptible soybean plants, but also in how severely they colonize the stems of these soybean plants. In our experiments, genotype A and genotype B did not differ consistently in their ability to cause internal stem discoloration. The two genotypes of C. gregata can be distinguished based on how severely they colonize stems of genotype A-susceptible soybean. Thus, a BSR resistance screening method, which relies on assessment of stem colonization by C. gregata, works only for screening soybean lines resistance to genotype A. In light of these results, it is important to distinguish soybean resistance to genotype A versus genotype B of C. gregata. Whether genotype B causes yield loss and whether soybean plants can be distinguished as resistant or susceptible to genotype B needs to be investigated.

Soybean Stem Colonization by Genotypes A and B of Cadophora gregata Increases with Increasing Population Densities of Heterodera glycines.

Growth chamber experiments were conducted to investigate whether parasitism by increasing population densities of Heterodera glycines, the soybean cyst nematode, increases the incidence and severity of stem colonization by the aggressive genotype A and the mild genotype B of Cadophora gregata (Phialophora gregata), causal agents of brown stem rot of soybeans. Soybean genotypes with three combinations of resistance and susceptibility to H. glycines and genotype A of C. gregata were inoculated with each genotype of C. gregata alone or each genotype with two population densities of H. glycines eggs, 1,500 or 10,000 per 100 cm3 of soil. Stems of two H. glycines-susceptible soybeans were more colonized by both aggressive and mild genotypes of C. gregata in the presence of high than in the presence of low H. glycines population density.

A New Greenhouse Method to Assay Soybean Resistance to Brown Stem Rot.

Greenhouse, growth chamber, and field experiments were conducted to develop a method to assess resistance of soybeans to Cadophora gregata (Phialophora gregata), causal agent of brown stem rot (BSR). In the new method, C. gregata is introduced at the base of the stems of 2-week-old soybeans, and the presence of the fungus is assessed in the tips of the stems 5 weeks later. To test the effectiveness of the method, two populations of soybeans and 10 checks were inoculated at the stem base and then assayed for fungal colonization of the stem tips, percentage of symptomatic leaflets, and percent internal stem length discolored. The lines also were planted in naturally infested fields to assess for percent internal stem length discolored, and were tested for the presence/absence of a BSR-resistant molecular marker. Greenhouse, field, and molecular marker data were compared. Linear regression analysis suggested that percentage of plants with colonized stem tips explained 41 to 64% of the variability (P < 0.0001) in percent stem length discolored in the field and 58 to 85% of the variability (P < 0.0001) in molecular marker data for BSR resistance. Percent stem length discolored assessed in the greenhouse had the lowest correlation with percent stem length discolored in the field and with the molecular marker. Of three incubation temperatures tested, 22°C was the most conducive for distinguishing resistant/susceptible soybeans using the colonization method.

Resistance to Phialophora gregata Is Expressed in the Stems of Resistant Soybeans.

Growth chamber experiments were conducted to determine if resistance to Phialophora gregata, the causal agent of brown stem rot (BSR) of soybean, is expressed in the stems of resistant soybean genotypes. Upon introduction of the pathogen into the base of stems of 2-week-old seedlings, the fungus advanced with the growing tips of plants of susceptible genotypes but lagged behind in resistant genotypes. Five weeks after introduction of the pathogen, both mean percent stem length colonized by P. gregata and mean percentage of symptomatic trifoliate leaflets were significantly less for resistant than for susceptible genotypes. These results indicate that resistance can be expressed in the stems of resistant soybean plants and suggest that stem inoculation methods may be useful for assessing resistance to P. gregata. Also, in our experiments, internal stem discoloration was not as useful as colonization and foliar symptoms in discriminating resistant from susceptible genotypes.

Heterodera glycines Infection Increases Incidence and Severity of Brown Stem Rot in Both Resistant and Susceptible Soybean.

Growth chamber experiments were conducted to investigate whether parasitism by Heterodera glycines, the soybean cyst nematode, increases incidence and severity of brown stem rot (BSR) of soybean, caused by Phialophora gregata, in both resistant and susceptible soybean cultivars. Soybean genotypes with various combinations of resistance and susceptibility to both pathogens were inoculated with P. gregata alone or P. gregata plus H. glycines. In most tests of H. glycines-susceptible genotypes, incidence and severity of internal stem discoloration, characteristic of BSR, was greater in the presence than in the absence of H. glycines, regardless of susceptibility or resistance to BSR. There was less of an increasing effect of H. glycines on stem symptoms in genotypes resistant to both BSR and H. glycines; however, P. gregata colonization of these genotypes was increased. Stems of both a BSR-resistant and a BSR-susceptible genotype were colonized earlier by P. gregata in the presence than in the absence of H. glycines. Our findings indicate that H. glycines can increase the incidence and severity of BSR in soybean regardless of resistance or susceptibility to either pathogen.

Genetic mapping of telomeric DNA sequences in the maize pathogen Cochliobolus heterostrophus.

Numerous polymorphic bands were observed when genomic DNA of Cochliobolus heterostrophus was cut with restriction enzymes and hybridized with a telomere-specific probe. Bal31 digestion demonstrated that the majority of the bands were from the chromosome termini and thus constitute telomeres. However, numerous hybridizing sequences in the strain B30.A3.R.45 were Bal31 exonuclease-insensitive and thus located at least 600 bases internal to the chromosome ends. Segregation analysis of the polymorphic bands identified nine telomeres; seven were linked to other markers and thus permitted the proper orientation of linkage groups with respect to their chromosome ends. Three internal, telomere-homologous sequences, at least one of which may be subtelomeric, were also mapped.

Identification, characterization, and mapping of Ecm1, a locus affecting extracellular matrix production and lesion size in Cochliobolus heterostrophus.

A mutant of Cochliobolus heterostrophus lacking the outer layer of extracellular matrix around its germ tubes and hyphae was obtained by mutagenizing protoplasts. The mutant not only lacks the outer matrix, but also produces much smaller lesions on corn leaves than nonmutant strains; the area of mutant lesions averages 0.6 mm2 compared with 5.8 mm2 for nonmutant lesions. Genetic analysis demonstrated that the failure to produce the outer matrix cosegregates with the reduced lesion size, indicating that the two traits are controlled by the same locus, designated Ecm1 (Extracellular Matrix Deficient). The mutant retains normal growth on media and normal abilities to germinate, form appressoria, and penetrate corn leaves. This indicates that the outer matrix is not necessary for infection prior to entrance of the fungus into the leaf. It also indicates that the pathogenicity defect in this mutant is manifested after penetration. To facilitate future tests of whether the pathogenicity defect is caused by the lack of the outer matrix, Ecm1 was mapped. Seven markers linked to Ecm1 were found by analysis of amplified fragment length polymorphisms. Ecm1 maps to chromosome 4; the closest markers to Ecm1 are 5 cM distant, which is estimated to represent about 115 kb.

A reciprocal translocation and possible insertion(s) tightly associated with host-specific virulence in Cochliobolus heterostrophus.

A reciprocal translocation and one or more apparent insertions are shown to be tightly associated with Tox1, a locus controlling T-toxin production and host-selective virulence in race T of the maize pathogen Cochliobolus heterostrophus. Chromosome arrangements were examined by separating chromosomal DNAs of a variety of Tox+ and Tox− strains by pulsed-field gel electrophoresis and hybridizing with probes known to detect RFLPs genetically linked to Tox1. The existence of the translocation was demonstrated by chromosome hybridization patterns; the existence of the putative insertion(s) was deduced from chromosome migration rates. Both differences in chromosome arrangement were detected between 8 Tox+ and 8 Tox− near-isogenic laboratory strains, suggesting that the differences are tightly linked to Tox1. The reciprocal translocation was also detected between all 7 Tox+ and 8 Tox− field isolates examined, suggesting that the translocation is common in natural populations. The field isolates may also differ by the insertion(s); however, numerous additional chromosome size polymorphisms in the field isolates prevented a firm conclusion. The tight association of the translocation and insertion(s) with T-toxin production suggests that chromosome rearrangements may have been involved in the evolution of race T and Tox1. These genomic differences may be causally related to the previously reported reduced fitness of race T relative to race O on N-cytoplasm maize. Key words : chromosome rearrangement, southern corn leaf blight, pulsed-field electrophoresis, T-toxin, evolution.

A restriction fragment length polymorphism map and electrophoretic karyotype of the fungal maize pathogen Cochliobolus heterostrophus.

A restriction fragment length polymorphism (RFLP) map has been constructed of the nuclear genome of the plant pathogenic ascomycete Cochliobolus heterostrophus. The segregation of 128 RFLP and 4 phenotypic markers was analyzed among 91 random progeny of a single cross; linkages were detected among 126 of the markers. The intact chromosomal DNAs of the parents and certain progeny were separated using pulsed field gel electrophoresis and hybridized with probes used to detect the RFLPs. In this way, 125 markers were assigned to specific chromosomes and linkages among 120 of the markers were confirmed. These linkages totalled 941 centimorgans (cM). Several RFLPs and a reciprocal translocation were identified tightly linked to Tox1, a locus controlling host-specific virulence. Other differences in chromosome arrangement between the parents were also detected. Fourteen gaps of at least 40 cM were identified between linkage groups on the same chromosomes; the total map length was therefore estimated to be, at a minimum, 1501 cM. Fifteen A chromosomes ranging from about 1.3 megabases (Mb) to about 3.7 Mb were identified; one of the strains also has an apparent B chromosome. This chromosome appears to be completely dispensable; in some progeny, all of 15 markers that mapped to this chromosome were absent. The total genome size was estimated to be roughly 35 Mb. Based on these estimates of map length and physical genome size, the average kb/cM ratio in this cross was calculated to be approximately 23. This low ratio of physical length to map distance should make this RFLP map a useful tool for cloning genes.