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Dep. of Agronomy and Plant Pathology, Univ. of Kentucky, Lexington, KY 40546-0312
* Corresponding author (dtekrony{at}uky.edu)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: DON, deoxynivalenol • dpi, days postinoculation • FHB, Fusarium head blight • PI, point inoculation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Spread of scab within a spike has been recognized as a relatively reliable index of Type II resistance in cultivars (Bai et al., 1999). Histological studies of pathogen spread in the spikelet provide evidence that the path of infection follows the vascular tissue, with most rapid growth in the longitudinal direction with slower transverse growth (Schroeder and Christensen, 1963; Ribichich et al., 2000). Susceptible wheat cultivars infected with F. graminearum have greater indices of spread in the rachis when compared with resistant cultivars. Ribichich et al. (2000) reported thickening of parenchyma walls and occluded but intact phloem in an infected resistant cultivar, and no defensive reaction with occluded and destroyed phloem in a susceptible cultivar. Mesterhazy (1995) noted variation in fungal movement is more stable in resistant varieties. In describing the relationship between resistance and movement, Savard et al. (2000) found DON to be localized primarily downward from the point of inoculation in a susceptible cultivar, but failed to note the presence or absence of F. graminearum or the relationship to the visual rating of spikelet infection.
Pandeya and Sinha (1998) reported a poor relationship between visual FHB disease rating of spikelets in the greenhouse and presence of DON in wheat seeds. Schroeder and Christensen (1963) proposed that the clogging of vascular tissue in the rachis above the point of inoculation of infected spikes caused the head to ripen prematurely, so that even grains not directly infected were shriveled because of a shortage of water and nutrients. Snijders and Krechting (1992) reported this premature white-head symptom led to higher variation in estimates of DON and ergosterol [(22E)-Ergosta-5,7,22-trien-3beta-ol] contents. Results derived from the visual rating system of spikelet infection for wheat lines regarded as either resistant or susceptible may therefore be inaccurate, and alternative methods of measuring pathogen spread in the spike have been proposed, including spread in the rachis (Wang, 1982).
Questions remain regarding the movement of F. graminearum hyphae in inoculated wheat spikes and its relationship to cultivar resistance. The objectives of this study were to determine (i) the levels and movement of F. graminearum into floral components following PI in the greenhouse, and (ii) the relationship between the visual rating system of spikelet infection (severity) in the greenhouse to seed infection by F. graminearum in the same spikes in resistant and susceptible cultivars.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-[2-(4-chlorophenyl)ethyl]--(1,1-dimethyl-ethyl)-1H-1,2,4-triazole-1-ethanol} fungicide and held in a vernalization chamber at 4°C and constant relative humidity for approximately 8 wk. Vernalized seedlings were sown in soil in 275-cm3 pots (1 seedling pot–1) in the greenhouse and plants were grown at 24/18°C on a 16-/8-h day/night regime. Two applications of liquid fertilizer (20–20–20 N–P–K) were applied to pots 1 wk after planting and before heading. Elemental sulfur was applied for control of powdery mildew and imidacloprid {1-[(6-chloro-3-pyridinyl)methyl]-N-nitro-imidazolidinimine} was applied at recommended rates for aphid control. Inoculum for screening for Type II resistance was prepared from a composite of 12 different isolates of F. graminearum. Ten isolates were obtained from scabby infected wheat seed found locally in Kentucky, and two were from Virginia (Carl Griffey, Virginia Tech University, 2000, personal communication). A single spore culture was derived from each field isolate and maintained on acidified potato dextrose agar (0.1% lactic acid). Macroconidial cultures were initiated by placing two mycelial plugs from the individual cultures of F. graminearum in separate flasks of carboxymethocellulose liquid media (Cappellini and Peterson, 1965). The flasks were placed on a shaker (115 rpm) for 2 wk at 24°C. Macroconidia were collected by filtering cultures through a 3.0-µm Millipore filter system. Following filtration, macroconidia were resuspended in a small amount of sterile water (2–3 mL) and streaked on mung bean agar plates (20 g mung beans + agar) and incubated at 20 to 25°C with a 12-h photoperiod for 7 d. Plates were washed with 5 to 10 mL sterile water, and the combined suspension from all isolates was calibrated to 600000 spores mL–1 with a hemacytometer. Subsequent transfers were continued using mung bean agar plates throughout the duration of the experiment.
At anthesis, a floret of a middle spikelet for each entry was marked with a permanent nontoxic pen. Two microliters of a macroconidial suspension (
1000 spores mL–1) was pipetted between the lemma and palea of each marked floret. After inoculation, plants were moved to a humidification chamber and continuously misted for 72 h at high temperature (25–35°C) and 100% relative humidity as described by Bai et al. (2001). Pots were then moved to greenhouse benches, and inoculated spikes were visually rated for disease incidence and severity at 21 and 28 dpi (days postinoculation) (Stack and McMullen, 1995). Bleached and/or discolored spikelets were recorded at each evaluation date, and the severity of infection was expressed as a percentage of the total number of spikelets on each spike (Stack and McMullen, 1995).
2000 Experiment
Three experimental wheat breeding lines, SC 921299 (SC), GA 89482-E7 (GA), and VA96W-326 (VA), characterized as being susceptible, and two cultivars, Roane and Ernie, characterized as resistant to FHB (based on previous evaluation of resistance levels; Hall and Van Sanford, 2003), were selected from the Uniform Southern FHB Type II Screening Nursery. Vernalized seedlings of the five genotypes were planted in the greenhouse in a completely randomized design on 11 Oct. 1999, and eight spikes (replications) were point inoculated at anthesis (Feekes 10.1). At seed maturity (Feekes 11.4, 120 g kg–1 moisture content), the spikes of each breeding line and cultivar were hand harvested, and the lowest right floret of all spikelets on the spike was dissected into individual floral components (rachis, glume, lemma, palea, and seed) and identified by spikelet location. Spikelets were numbered in relation to their relative position to the inoculated spikelet (0), positive numbers representing the spikelets above the PI and negative numbers representing the spikelets below the PI (i.e., +1 and –1 being the spikelets directly above and below the PI, respectively).
Following pretreatment with NaOCl (1% available chlorine) for 2 to 5 min (Haflon-Meiri et al., 1979), floral components were plated on modified PDA agar (39.5 g PDA + 1.5 g pentachloronitrobenzene + 0.5 g chloramphenicol + 1 L water) (Fauzi and Paulitz, 1994), grown in light at 25°C for 14 d, and evaluated for F. graminearum infection. Infection by F. graminearum was confirmed by random microscopic comparison of the cultural morphology of the isolated fungus (Tuite et al., 1990) to the original inoculum source fungus on the selective media. Several representative colonies from infected seeds were speciated on carnation (Dianthus caryophyllus L.) leaf agar according to Nelson et al. (1983) to assist in F. graminearum colony identification. The mean infection of individual components of each floret for each genotype was determined by averaging the number of infected components across the total number of spikelets in eight spikes for that genotype. Spikelets were numbered in relation to their relative position as described above.
2001 Experiment
Similar to 2000, Type II screening was performed on 10 plants each of the three breeding lines (SC, GA, and VA) and Roane. Vernalized seedlings were transplanted to the greenhouse on 27 Jan. 2001 and flowers were point inoculated at anthesis and rated for spikelet infection as in 2000. At maturity, 10 spikes (replications) for each genotype were hand harvested and held at 10°C until the seed and rachis were plated by spikelet location as in 2000.
Five plants of all wheat cultivars and breeding lines in the Uniform Northern (n = 49) and Southern (n = 29) FHB Screening Nurseries were grown in the greenhouse in a completely randomized design as described above and point inoculated at anthesis. Individual and average FHB spikelet infection (severity) across the five spikes was determined for each genotype at 21 dpi and the genotypes were grouped into the three categories based on levels of spikelet infecton; resistant (<25% spikelet infection), susceptible (50 to 75% spikelet infection), and highly susceptible (>75% spikelet infection). Within each group, seeds from all spikes were evaluated for F. graminearum seed infection by spikelet location (as in 2000) and the mean seed infection above (+) and below (–) the PI was determined for each resistant/susceptible genotype group. The results of plating for seed infection were related to the visual greenhouse ratings of FHB spikelet infection (severity), and trends in movement were analyzed.
All genotypes from the Uniform FHB Northern and Southern nurseries were established in a randomized complete block design with two replications in the FHB field nursery in 2001. Corn (Zea mays L.) seed infected with F. graminearum was used as an inoculum source and plots were irrigated during two periods daily as described by Argyris et al. (2003) to provide near epidemic conditions. Mean incidence and head severity of FHB in the field plant population was calculated according to the methods of Stewart (2003). Following harvest, the percentage of scabby seed was calculated as a percentage of a 200-seed sample showing visibly Fusarium damaged (shriveled, tombstone) seeds. Fifty seeds from the harvested sample were also plated for F. graminearum as described above. Average disease incidence and severity in the field and the percentage of scabby seed from field harvested seed samples were compared with average laboratory F. graminearum seed infection from field-harvested seed and to prior greenhouse evaluations of average FHB spikelet infection following point inocculation for the same genotypes. Correlation analysis of field and greenhouse data was performed using PROC CORR of SAS (Cary, NC).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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8%) to visual ratings in the greenhouse. Average spikelet infection for the resistant Roane was also low (8%) in the greenhouse; however, much higher average levels of F. graminearum occurred for all plated floral components (rachis and seed infection levels were 40 and 29%, respectively).
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When the five floral components of only the inoculated floret (0 location) were evaluated, all components for all genotypes had 100% infection with F. graminearum, except the palea of Ernie and Roane and the glume of Roane (Fig. 1) . Lower levels of F. graminearum infection (primarily in the rachis) occurred at all other spikelet locations for Ernie, moving five spikelets below (–1 to –5) the PI, but only to two spikelets above the PI (Fig. 1A). Rachis infection in Roane ranged from 50 to 63% for all spikelet locations below the PI, but declined sharply to low levels by the third spikelet above this point (0–38%, Fig. 1B). Similar trends of higher infection at all spikelets below the PI than above were shown for the other components of Roane and for all components for the VA and SC genotypes, but infection dropped off sharply above this point (Fig. 1C, VA not shown).
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2001 Experiment
Visual estimates of FHB spikelet infection in the greenhouse ranged from 8% (Roane) to 74% (VA) (Table 1), which was similar to 2000 for all genotypes except SC, which was 50 percentage points lower. Rachis infection with F. graminearum in the laboratory always exceeded seed infection; however, the differences between the two components were similar to 2000, ranging from 2 (SC) to 10 (Roane) percentage points. Seed infection with F. graminearum ranged from higher (Roane, SC) to lower (GA, VA) than visual ratings of FHB spikelet infection in the greenhouse.
Spikes produced in the greenhouse in 2001 were smaller and had fewer spikelets than in 2000; however, movement trends of F. graminearum infection in the seeds and rachis following PI were similar to 2000, except for GA (data not shown). As in 2000, higher levels of F. graminearum infection occurred in the seed and rachis below the PI in Roane, VA, and SC, while infection declined to low levels above the PI. In contrast to 2000, seed infection for GA was also high below the PI, but declined to low levels above the PI.
Individual heads of many genotypes in the Northern and Southern Uniform FHB Nurseries showed low levels (
10%) of FHB spikelet infection (severity) (Fig. 2A and 2B
, respectively). The level of F. graminearum infection of seeds from these same spikelets in both nurseries, however, ranged from 0 to 100% (mean = 25%). Likewise, those individual heads which showed 100% FHB severity in the greenhouse averaged from 4 to 92% (mean = 56%) seed infection in the laboratory. Even those spikes showing moderate severity levels (40–60%) of infection in the greenhouse had a wide range in seed infection (5–90%). Although the data were variable, correlations between visual ratings of FHB severity of individual heads in the greenhouse and seed infection from the same heads were significant (r = 0.53**, r = 0.45**, P < 0.01) in both the Northern and Southern Uniform FHB nurseries, respectively).
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When the spikes of all genotypes in the Uniform Northern and Southern FHB nurseries were grouped by resistance and susceptibility to FHB infection, similar trends in F. graminearum seed infection and movement in the spike was observed for each group (Fig. 3)
. For those highly susceptible genotypes (75–100% spikelet infection in the greenhouse), 100% seed infection was shown at the PI and remained near this level down the spike (Fig. 3A). High average levels of infection (92%) were shown at only the first spikelet (+1) above the PI and declined rapidly at the next five upper spikelet locations to 11% infection at +5. Those genotypes classified as susceptible (50–75% spikelet infection) also exhibited much higher seed infection (65–90%) at spikelets below the PI than for those spikelets above the PI (Fig. 3B). As expected, those genotypes classified as resistant to Type II FHB spikelet infection (25% spikelet infection in the greenhouse) had lower levels of seed infection in the laboratory (Fig. 3C). Seed infection at the PI was 77% for resistant genotypes and gradually declined to 34% at the –5 spikelet below the PI. The seed infection above (+) the PI declined sharply at the first spikelet to 29% and continued to decline up the spike.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Genotypic response to F. graminearum seed infection and movement of the fungus within the spike was divided into two categories in 2000 and 2001; localization around the PI (Ernie, Roane), movement primarily downward from the PI (VA, SC, GA, 2001). Movement of F. graminearum both up and down the spike from the PI only occurred for the susceptible GA genotype in 2000 (Fig. 1). Thus, for a resistant (Ernie) or moderately resistant (Roane) cultivar, the fungus was primarily localized around the PI. When resistant genotypes (<25% spikelet infection) in the Uniform FHB nurseries were grouped, F. graminearum seed infecton in the spike was also less severe basal and distal to the PI compared with susceptible genotypes (Fig. 3). Additionally, movement of F. graminearum in the rachis usually exceeded seed infection by several spikelets in the upward direction in inoculated spikes, but did not spread into the floral components of the same spikelets adjacent to the site of infection (Fig. 1). These results would imply the existence of mechanisms limiting the spread of the pathogen in the rachis as suggested by Bai and Shaner (1996), who speculated that resistant cultivars may have some substance that suppresses growth of the mycelium in the spike. These results also support the findings of Ribichich et al. (2000), who noted anatomical differences between resistant and susceptible cultivars to movement of F. graminearum in the wheat spike.
Visual symptoms of FHB greenhouse spikelet infection were evident early in development, and were generally more severe in greenhouse inoculated spikes of susceptible (GA, SC, VA) genotypes compared with the resistant cultivar Ernie and the moderately resistant cultivar Roane (Table 1). Visual estimates as high as 100% FHB head severity were recorded in individual spikes of many genotypes of the Uniform Northern and Southern FHB nurseries despite seed infection levels in the same spikes as low as 32% (Fig. 2A, B). In contrast, many genotypes with low visual levels of FHB head severity (<10%) in individual spikes had seed infection ranging from 0 to 100%. In susceptible genotypes, movement occurred primarily down the spike from the PI, which resulted in higher levels of seed infection below the PI (Fig. 1, 3). The primary reason for the poor relationship between seed infection and visual rating of FHB spikelet infection in individual spikes was likely due to the lack of movement of F. graminearum to seed and other floral components above the point of inoculation and the sudden desiccation (visually rated as infected) of the upper portion of spikes in the greenhouse. This resulted in visual estimates of disease severity that were higher in susceptible genotypes when the desiccation was mistaken for FHB head infection, compared with resistant lines where disease expression and variation in fungal movement was more stable. Snijders and Krechting (1992) also reported that infection spread to the rachis from spikelets of infected plants causing the premature white-heads symptom, which led to higher variation in estimates of DON and ergosterol contents for visually rated spikelet infection. Although DON was not measured in individual seeds in our study, Savard et al. (2000) reported high levels of DON at all spikelets below the PI of a middle spikelet in a susceptible cultivar, but little DON accumulation above the PI. This supports our conclusions of greater F. graminearum movement below the PI and would question the accuracy of greenhouse disease ratings when visually evaluating genotypes for FHB Type II resistance, in agreement with Hall and Van Sanford (2003).
Spikelet infection among individual spikes often ranged from 0 to 100% within a genotype (Fig. 2A, B). This could be attributed to heterogeneity of the breeding lines or possible mechanical contamination. Thus, mean visual ratings of FHB head infection across a number of individual spikes would appear to provide a more accurate assessment of Type II resistance. This assumption was supported by a significant (P = 0.05) linear relationship between the mean of FHB head severity and F. graminearum seed infection from the same heads for the both the Northern and Southern nurseries (Fig. 2C, D). Only when the greenhouse ratings of visual FHB spikelet infection (severity) remained 30% did the F. graminearum seed infection of individual spikelets remain below 30% (Fig. 2A, B). As seed infection exceeded 30%, the relationship between seed infection and the visual rating of the same spikes became highly variable in individual spikes for genotypes rated as resistant and susceptible. Mesterhazy (1995) has suggested that Type II resistance and resistance to seed infection are independent resistance mechanisms.
Individual spikes selected for resistance (10% FHB infection) based on the visual rating had up to 100% seed infection (Fig. 2), while spikes from genotypes regarded as highly susceptible (100% FHB infection) had as little as 30% seed infection. Thus, the variability in the visual rating system of individual spikes could have negative implications in the selection of genotypes for Type II resistance, allowing some susceptible individuals to persist, and ignoring potentially moderately resistant individuals or individuals with a different resistance mechanism (Mesterhazy, 1995). For this reason, a combination of both the greenhouse rating of FHB infection and the assessment of laboratory seed infection would allow for a more definitive selection of individuals showing Type II resistance. Likewise, setting an arbitrary limit of 30% on both greenhouse and seed infection may more frequently identify promising genotypes which show potential resistance.
Fusarium graminearum infection of seed harvested in the field from the Uniform Northern and Southern FHB field nurseries in 2001 was severe (mean > 90%) in all genotypes. Greenhouse estimates of Type II resistance (visual FHB head infection following PI) was much lower than mean FHB head severity of the same genotypes in the field environment and severity in the two environments was not highly correlated (r = 0.50). This was clearly illustrated in Roane, where greenhouse PIs resulted in visual FHB spikelet and seed infection of 8 and 9%, respectively (Table 1). The ratings for visual FHB spikelet infection were much higher in the field (33%). Likewise, the seed produced in the field environment had a high percentage of scabby seed (49%) and high levels of F. graminearum seed infection (94%). If resistance to spread of the pathogen in the spike is indeed stable, and less variable in resistant genotypes, the poor field performance may be attributable to Type I infections overwhelming any benefit of Type II resistance. Thus, Type II resistance would appear to be less effective in reducing seed infection under the severe FHB epidemic conditions encountered in the field nurseries in 2001. It may be of more value when the supply of primary inoculum is limited. The inability to distinguish between Type I and Type II resistance renders the benefits of Type II resistance observed in the greenhouse difficult to assess in the field, and as Bai and Shaner (1994) noted, many variables not directly related to host resistance can affect primary infection, complicating the process.
These studies showed a poor relationship between visual ratings of FHB spikelet infection (severity) following PI of a middle spikelet in the greenhouse and F. graminearum seed infection in the same spikes in the laboratory across five genotypes that have a range of Type II resistance to FHB. Greenhouse spikelet infection ranged from 5 to 100% for individual heads of resistant and susceptible genotypes, respectively, with a wide but unrelated range in seed infection in heads from the same genotypes. Fusarium graminearum movement within the spike following PI was higher in the rachis tissue of each spikelet than the seed and other floral components. In susceptible genotypes, F. graminearum tended to move to lower spikelets below the point of inoculation more frequently than upper spikelets. Field estimates of FHB head severity were also poorly related to F. graminearum infection of the harvested seed across a wide range of genotypes. For the cultivars studied, it appears that Type II resistance as measured in the greenhouse or the field provides an unreliable estimate of seed infection by F. graminearum of the same spikelets.
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ACKNOWLEDGMENTS |
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Received for publication January 6, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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