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Published online 30 July 2007
Published in Crop Sci 47:1661-1671 (2007)
© 2007 Crop Science Society of America
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FORAGE & GRAZINGLANDS

Potato Leafhopper Injury and Fusarium Crown Rot Effects on Three Alfalfa Populations

J. J. Arissa, L. H. Rhodesa, R. M. Sulcb,* and R. B. Hammondc

a Dep. of Plant Pathology, Ohio State Univ., Columbus, OH 43210
b Dep. of Horticulture and Crop Science, Ohio State Univ., Columbus, OH 43210
c Dep. of Entomology, Ohio State Univ., Wooster, OH 44691. Salary and research support provided in part by state and federal funds appropriated to the Ohio Agric. Res. and Dev. Ctr. (OARDC) and The Ohio State Univ. including an Interdisciplinary Team Research Grant from the OARDC Research Enhancement Competitive Grants Program, and by a gift from Forage Genetics, International

* Corresponding author (sulc.2{at}osu.edu).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Evidence suggests that homopterous insects and crown-rotting Fusarium species interact to impose stresses affecting alfalfa (Medicago sativa L.) persistence; however, few experiments quantify the combined effects of those two stressors on alfalfa. Our objective was to investigate the effects of Fusarium crown rot and potato leafhopper (Empoasca fabae Harris), alone and in combination, on three alfalfa populations differing in resistance to potato leafhopper and Fusarium crown rot. Treatments were Fusarium-inoculated plus leafhopper-infested, only leafhopper-infested, only Fusarium-inoculated, and uninoculated–uninfested in 35-wk experiments. Potato leafhoppers were introduced onto plants in greenhouse cages either before or after inoculation with a known crown-rotting isolate of Fusarium oxysporum Schlect. There were few Fusarium x potato leafhopper or Fusarium x potato leafhopper x population interactions, indicating that overall, the effects of Fusarium and potato leafhopper were additive. Both Fusarium and potato leafhopper injury caused stand losses, and the combined effects of Fusarium and potato leafhopper, under two different inoculation plus infestation sequences, were essentially the same. In the presence of both Fusarium and potato leafhopper, the population having resistance to both organisms had a three- to sevenfold increase in plant survival and a three- to 4.6-fold increase in yield compared with the population lacking resistance to either organism.

Abbreviations: APDA, acidified potato dextrose agar • Control, uninoculated, uninfested • FUS+, Fusarium inoculated • FUS+PLH+, Fusarium inoculated plus potato leafhopper infested • PLH+, potato leafhopper infested


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MODERN ALFALFA (Medicago sativa L.) cultivars typically possess resistance to prevalent diseases and insects, yet stand persistence is often poor. Vague, ill-defined terms such as "stand decline" and "lack of persistence" are applied to the premature degeneration of alfalfa stands when a single causal agent cannot be identified. Infection by crown-rotting organisms may contribute to reduced stand persistence. The term crown rot complex of alfalfa and other forage legumes refers to the many organisms acting in concert that result in the destruction of the crown and portions of the roots. Fungi, bacteria, and nematodes all may play a role in the crown rot complex. Rhizoctonia solani, Phoma spp., Colletotrichum spp., and Pythium spp. are a few of the pathogens that may be associated with crown rot of alfalfa; however, Fusarium species stand out as the organisms most commonly isolated from rotted alfalfa crowns and most frequently implicated as causal agents of crown rot (Leath et al., 1971).

Fusarium crown rot develops slowly, often during the course of several years, and may cause or contribute to plant death. Symptoms begin with dark brown discoloration of crown tissue that advances longitudinally through the crown and into the taproot. The infected tissue eventually becomes necrotic, and the affected plant is often stunted, asymmetrical, and if unable to recover or compensate, ultimately dies. While Fusarium species may enter and infect alfalfa roots directly, inducing a root rot, the sites of infection that induce crown rot are frequently old stems and crown wounds (Wilcoxson et al., 1977; Richard et al., 1980; Turner and Van Alfen, 1983).

Fusarium crown rot is often reported to be favored by biotic and abiotic stresses, and conversely, the effects of such stresses have been reported to be more severe when plants are affected by Fusarium crown rot. Mechanical injury and the associated stress typical of that caused by harvesting procedures have been shown to favor the development of Fusarium crown rot (O'Rourke and Millar, 1966). Decreased winter hardiness has been found in association with Fusarium crown rot (Wilcoxson et al., 1977), and winterkill in alfalfa is more frequent in plants infected by Fusarium species (Hwang and Flores, 1987; Richard et al., 1982). Because the incidence and severity of Fusarium crown rot in an alfalfa stand increases with stand age (Hawn, 1958), the cumulative or synergistic effects of biotic and abiotic stressors with time result in a "cumulative stress load" that affects alfalfa persistence (Leath, 1989). However, Fusarium isolates associated with field-rotted plants may cause little stunting and rot when inoculated into healthy plants in controlled inoculation experiments, providing evidence that Fusarium crown rot is often a stress-induced disease or that Fusarium is only one component of the disease complex (Hancock, 1985; MacDonald, 1955).

The effects of potato leafhopper (Empoasca fabae Harris) feeding on alfalfa can be severe. In the north-central regions of the USA, potato leafhopper infestations usually occur before the second cutting and may continue through the end of summer if left uncontrolled (Manglitz and Ratcliffe, 1988). The most common symptoms of potato leafhopper feeding in alfalfa are yellowing of leaves and stunted growth. Dry matter yield and forage quality may be reduced not only in the harvest cycle in which the infestation occurred but in subsequent harvests as well (Kindler et al., 1973). Potato leafhopper infestation may cause delayed budding (Wilson et al., 1979) and flowering (Kindler et al., 1973). Control of potato leafhopper has been largely dependent on chemical insecticide applications until the recent commercial release of glandular-haired, potato leafhopper resistant varieties. The glandular-haired trait confers a degree of resistance to potato leafhopper (Shade et al., 1979; Ranger and Hower, 2001, 2002; Ranger et al. (2004, 2005). The initial germplasm lines of the current glandular-haired releases consisted of selections of M. sativa, M. sativa ssp. praefalcata, M. glutinosa, M. prostrata, and M. glandulosa (Elden and McCaslin, 1997). Seasonal forage yield for the first glandular-haired commercial releases ranged from 6% below to 15% above the yield of leafhopper-susceptible cultivars when leafhoppers were not controlled with insecticides (Sulc et al., 2001; Hansen et al., 2002). Recent trials in Iowa and Ohio have demonstrated that the best leafhopper-resistant cultivars currently available have reasonable yields ranging from 20 to 50% higher than those of susceptible check cultivars in the absence of insecticide treatment (Smith and Brummer, 2005; Sulc et al., 2005).

Certain insect feeding and injury has been shown to predispose alfalfa to infection by Fusarium species and favor the subsequent colonization of the crown and root regions in alfalfa. There is evidence that insects that either provide a chewing wound or feed via a stylet may have significant roles in Fusarium crown rot. Three-cornered alfalfa hopper (Spissistilus festinus Say) feeding was found to increase the severity of Fusarium crown rot (Moellenbeck et al., 1992). Similarly, development of Fusarium crown rot was favored by feeding of pea aphid (Acrythosiphon pisum Harris) and potato leafhopper, and ultimately resulted in reduced alfalfa persistence (Leath and Byers, 1977).

Many investigators have established the role of Fusarium species in crown rot of alfalfa; however, the relationship of Fusarium crown rot to alfalfa persistence and the factors that contribute to its incidence and severity are important yet relatively unexplored topics. There are few previous investigations on Fusarium crown rot in alfalfa where controlled and quantified inoculation techniques have been used. While much is known about the effects of potato leafhopper on alfalfa, much less is definitively known about Fusarium crown rot, and the relationship between the two stresses remains largely undocumented. The prevalence of both crown-rotting Fusarium species and potato leafhopper in a typical alfalfa production field indicates the need for a more thorough understanding of any interactions between the three organisms. For this reason, greenhouse experiments were undertaken to determine the effect of potato leafhopper injury on the development and severity of Fusarium crown rot in alfalfa and to explore the effects of the order in which Fusarium infection and potato leafhopper stress are imposed.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Two experiments were conducted in the greenhouse. In each experiment, factors involved were alfalfa population (i.e., cultivar or experimental line), Fusarium oxysporum inoculation, and potato leafhopper infestation. The alfalfa populations differed in resistance to Fusarium and potato leafhopper. Treatments applied to alfalfa populations were uninoculated and uninfested (Control), Fusarium-inoculated (FUS+), potato leafhopper infested (PLH+), and Fusarium-inoculated plus potato leafhopper infested (FUS+PLH+). The resulting experiments were 3 x 2 x 2 full factorials (i.e., 12 treatments) with four replications per treatment in a completely randomized design. In Exp.1, potato leafhoppers were applied before Fusarium inoculation, while in Exp. 2, potato leafhoppers were applied after Fusarium inoculation. Each experiment was conducted twice.

Plant Populations
Three alfalfa populations were selected for these studies. Populations were provided by Forage Genetics International, La Crosse, WI. Two of the population selections (FG1-1 and FG2-1) were based on the identification of a persistence trait. Experimental data (Rhodes, 2000) identified a component of this persistence trait as Fusarium crown rot resistance. The FG1-1 population was a glandular-haired selection with potato leafhopper resistance. The FG2-1 population was a non-glandular-haired selection, lacking potato leafhopper resistance. Both FG1-1 and FG2-1 populations had undergone one cycle of selection for resistance to Fusarium crown rot. The third selected population was the cultivar ‘LegenDairy’; a non-glandular-haired cultivar lacking potato leafhopper resistance and identified as having reduced persistence when inoculated with cortical-rotting Fusarium isolates (Rhodes, 2000).

Plant Culture
Fifteen seeds were planted in 15-cm-diameter plastic pots in a sterilized planting medium consisting of a 1:1:1 (v/v/v) ratio of Crosby silt loam (fine, mixed, active, mesic Aeric Epiaqualfs), peat, and vermiculite. A solution of nutrients and lime was added before sterilization of the soil to adjust the nutrient level to 82 mg kg–1 P, 195 mg kg–1 K, 4690 mg kg–1 Ca, and 648 mg kg–1 Mg. A template was used for placement of the seed to ensure even spacing between plants. Scarified seeds were treated with a commercial preparation of Sinorhizobium meliloti to ensure nodulation. Seeds were germinated on a mist table. Seedlings were thinned to 12 plants pot–1 and then moved to a greenhouse bench ~2 wk after seedling emergence. Thus, the experimental unit consisted of one pot of 12 plants. In Exp. 1, each pot was placed in an individual leafhopper-proof cage 5 wk after planting. In Exp. 2, pots were placed in cages at ~10 wk after planting. Leafhopper cages consisted of glass panels on the top and three sides. The remaining side was covered with nylon fabric. Each cage was wholly contained with individual water and forced-air supply.

Potato Leafhopper Infestation
For both experiments, 15 greenhouse-reared adult potato leafhoppers, consisting of approximately 80% females, 4 to 8 d of age, were added to pots designated for the PLH+ treatment. At the conclusion of the infestation period, potato leafhoppers were removed using a modified handheld vacuum, and potato leafhopper adults and nymphs were killed by freezing and then counted. A potato leafhopper yellowing rating was given before potato leafhopper removal for all pots using the rating scale from Elden (1995): 1 = 0 to 20% leaves yellowing, 2 = 20 to 40% leaves yellowing, 3 = 40 to 60% leaves yellowing, 4 = 60 to 90% leaves yellowing, and 5 = leaves necrotic and stems wilted. In Exp. 1, potato leafhoppers were applied 5 wk after seeding and removed at 10 wk, immediately before the first harvest. In Exp. 2, potato leafhoppers were introduced onto regrowth 2 wk after the first harvest and removed immediately before the second harvest (15 wk after plant emergence).

Inoculum Preparation, Inoculation Technique and Harvests
An Ohio isolate of Fusarium oxysporum Schlect (isolate designation 9–1) obtained from rotted alfalfa crowns (Abdalla, 1986) was used for the experiments. A conidial suspension was prepared using 10 petri plates of acidified potato dextrose agar (APDA) (Difco Laboratories, Detroit, MI; 0.1% lactic acid by volume) streaked with F. oxysporum for uniform fungal growth. Plates were incubated for 9 d to ensure sufficient sporulation. Each plate was flooded with sterile water, the agar surface was scraped, and the remaining agar and mycelium was cut into approximately 100 cubes. The contents of the 10 plates were added to 250 mL of water, stirred, and stored at 4°C for 12 h. The suspension was then filtered through four layers of cheesecloth. The inoculum concentration was adjusted to 6 x 106 conidia mL–1 using a hemacytometer (Hausser Scientific, Horsham, PA). A control solution was prepared as above by using uncolonized APDA.

In both experiments, all treatments were harvested to a stubble height of 2.5 cm at 10 wk. Plants in treatments subjected to Fusarium inoculation (FUS+) were harvested using scissors dipped in the conidial suspension of F. oxysporum. Plants not subjected to Fusarium inoculation were harvested using scissors dipped in the control solution. Immediately following clipping, plants were spray-inoculated at a pressure of 173 kPa with 30 mL of either Fusarium conidial suspension or control solution. Pots were then placed into sealed plastic bags and kept in darkness for approximately 48 h to ensure maximum germination of Fusarium spores and subsequent colonization of stubble and crown tissue.

The first harvest was made at 10 wk, coinciding with the Fusarium inoculation. Five subsequent harvests were made at 35-d intervals, for a total of six harvests. All harvested plant material was dried in a forced-air oven at 60°C for 4 d. Dry matter yield per pot and number of harvestable stems (>15-cm length) per pot were recorded at each harvest. Plants per pot with apparent regrowth were counted immediately after each harvest and 2 wk after each harvest as a nondestructive determination of plant survival during the experiment.

Assessment of Crown Rot
After the sixth cutting, plants were lifted from the soil, washed, weighed, and rated for crown rot severity, crown symmetry, and the presence of secondary crowns. Crown rot ratings were based on a disease severity scale of the percentage of crown tissue affected: 1 = 0 to 20%, 2 = 21 to 40%, 3 = 41 to 60%, 4 = 61 to 80%, 5 = >81% affected. Crown symmetry scores were based on the visual appearance of overall crown symmetry compared with 360° aspect: 1 = 1 to 20% of crown nonsymmetrical, 2 = 21 to 40% of crown nonsymmetrical, 3 = 41 to 60% of crown nonsymmetrical, 4 = 61 to 80% of crown nonsymmetrical, and 5 = > 81% of crown nonsymmetrical. The presence or absence of secondary crowns was recorded for each surviving plant and the results were reported as the average percentage of survivors with secondary crowns.

A random sample of crown and root tissue of five plants from each treatment were surface sterilized in 0.5% NaClO for 1 min, rinsed with sterile water, cut into 10 segments, and plated onto two APDA plates. Plates were examined 4 d later for the presence of fungal growth morphologically similar to that of the F. oxysporum isolate used for inoculation.

Data Analysis
Analysis of variance was conducted for each experiment using the GLM procedure of SAS (SAS Institute, Cary, NC). In both experiments, there was a significant (P < 0.05) repetition (run) effect for several variables; however, there were few significant interactions between experiment repetitions and any treatment or treatment combination. Where those significant interactions occurred, the sum of squares associated with the interaction accounted for a low proportion of the total sum of squares. Thus, data were combined across repetitions of each experiment in the final analysis of variance. Means were compared using Fisher's protected LSD. An arcsine transformation was used for live plant percentage data for analyses but the untransformed data are presented for graphical representation. For those variables where two or more of the two-way interactions were significant or where the three-way interaction was significant, individual treatment means (12 treatment combinations) were compared using the LSD.


   RESULTS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Analyses of variance for both experiments showed main effect significance for most key dependent variables (Tables 1 and 2). The variables total dry matter yield (yield sum across all harvests), crown plus root fresh weight, percentage of live plants at 35 wk, crown rot rating, and crown symmetry rating were significant (P ≤ 0.05) for the main effects in both experiments. In general, there were significant potato leafhopper x population interactions for most variables, as might be expected with both the FG2-1 and LegenDairy populations being potato leafhopper susceptible and only the FG1-1 population having potato leafhopper resistance conferred by the glandular-haired trait. There were few significant Fusarium x potato leafhopper interactions or Fusarium x potato leafhopper x population interactions for any of the key dependent variables.


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  		Table 1. Experiment 1 overall analysis of variance; potato leafhoppers (PLH) applied before Fusarium inoculation of alfalfa (Medicago sativa L.) populations.

 

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  		Table 2. Experiment 2 overall analysis of variance; potato leafhoppers (PLH) applied after Fusarium inoculation of alfalfa (Medicago sativa L.) populations.

 
Plant Survival
In Exp. 1, analysis of variance for the percentage of surviving plants per pot at Week 35 (sixth harvest) indicated significant (P ≤ 0.05) effects of population, Fusarium, and potato leafhopper, as well as significant interactions for Fusarium x population and potato leafhopper x population (Table 1). There was no significant plant loss associated with either the PLH+, FUS+, or control (uninoculated, uninfested) treatments in the FG1-1 population; however, the FUS+PLH+ treatment resulted in 23.3% plant loss compared with the control at Week 35 (Fig. 1 , Exp. 1). In FG1-1, the combined stresses of Fusarium inoculation and potato leafhopper infestation resulted in what appeared to be a synergistic effect with regard to plant mortality; however, the lack of any significant Fusarium x potato leafhopper x population interaction indicates that overall, the nature of the effects of Fusarium and potato leafhopper injury were additive. In FG2-1, the FUS+PLH+ treatment resulted in 47% plant loss over the control treatment while the FUS+ treatment resulted in 17.3% plant loss compared with the control at 35 wk (Fig. 1, Exp. 1). The FG2-1 population had no significant difference in the average number of live plants per pot remaining at 35 wk between the FUS+PLH+ and the PLH+ treatments, as might be expected for a population selected for Fusarium crown rot resistance but lacking any potato leafhopper resistance. In LegenDairy, the FUS+PLH+ treatment resulted in 68.4% plant loss over the control, while the FUS+ treatment and the PLH+ treatment resulted in 30.5 and 28.4% plant loss, respectively (Fig. 1, Exp. 1).


Figure 1
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  		Figure 1. Proportion of live plants remaining at 35 wk in three alfalfa (Medicago sativa L.) populations (FG1-1, FG2-1, ‘LegenDairy’) exposed to four treatments in Exp. 1 and 2: not inoculated, not infested (Control); inoculated with Fusarium (FUS+); infested with potato leafhoppers (PLH+); and Fusarium inoculated plus potato leafhopper infested (FUS+PLH+). Means represented by bars with the same letter in each experiment are not significantly different (P = 0.05). Arcsine transformations were applied for data analysis; however, untransformed percentages are presented.

 
A similar pattern of surviving plants was observed in Exp. 2 (potato leafhopper infestation after Fusarium inoculation) (Fig. 1, Exp. 2). The FG1-1 population showed the greatest plant loss in the FUS+PLH+ treatment, with intermediate plant loss in the FUS+ and PLH+ treatments (Fig. 1, Exp. 2). In the second experiment, the percentage of live plants at 35 wk was lowest in the FUS+PLH+ treatments in the FG2-1 and LegenDairy populations (Fig. 1, Exp. 2). In the treatments where significant plant loss occurred (measured by lack of apparent regrowth following plant harvest), the majority of plant loss was observed between the first (Week 10) and third (Week 20) harvests, with plant loss largely ceasing by the third harvest and live plant counts remaining roughly static through the final harvest (Week 35) in all populations in both experiments (Fig. 2 and 3 ).


Figure 2
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  		Figure 2. Plant survival (proportion of plants showing visible regrowth) during 35 wk in three alfalfa (Medicago sativa L.) populations (FG1-1, FG2-1, ‘LegenDairy’) exposed to four treatments in Exp. 1: not inoculated, not infested (Control); inoculated with Fusarium (FUS+); infested with potato leafhoppers (PLH+); and Fusarium inoculated plus potato leafhopper infested (FUS+PLH+). Arcsine transformations were applied for data analysis; however, untransformed percentages are presented.

 

Figure 3
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  		Figure 3. Plant survival (proportion of plants showing visible regrowth) during 35 wk in three alfalfa (Medicago sativa L.) populations (FG1-1, FG2-1, ‘LegenDairy’) exposed to four treatments in Exp. 2: not inoculated, not infested (Control); inoculated with Fusarium (FUS+); infested with potato leafhoppers (PLH+); and Fusarium inoculated plus potato leafhopper infested (FUS+PLH+). Arcsine transformations were applied for data analysis; however, untransformed percentages are presented.

 
Yield
There were population, Fusarium, potato leafhopper, and potato leafhopper x population effects for total dry matter yield in both experiments. The lowest total dry matter yield (sum across all harvests) was observed in the inoculated, infested treatments (FUS+PLH+) in LegenDairy, although it was not significantly different from the PLH+ treatment in Exp. 1 (Tables 3 and 4). Dry matter yield was reduced in Exp. 1 in all potato leafhopper infested treatments compared with the control treatments, but the reduction was least in the potato leafhopper resistant FG1-1. No significant reduction in yield for any treatment was observed in the potato leafhopper resistant population (FG1-1) in Exp. 2. There was no reduction in yield associated with Fusarium inoculation alone (FUS+) in any population in either experiment compared with the control. Treatment differences for dry matter yield at individual harvests (data not shown) followed generally the same pattern to that observed for total dry matter yield. Stems per plant and stems per pot (data not shown) at most harvests showed relatively few differences between treatments within each population. Thus, differences in mass per stem (data not shown) accounted for the yield-per-pot differences observed among treatments. The only notable deviation in stem number was a significant reduction in stems per pot and stems per plant in the FUS+ treatment when compared with the control.


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  		Table 3. Response of three alfalfa (Medicago sativa L.) populations (FG1-1, FG2-1, ‘LegenDairy’) in Exp. 1 to four treatments: not inoculated, not infested (Control); inoculated with Fusarium (FUS+); infested with potato leafhoppers (PLH+); and Fusarium inoculated plus potato leafhopper infested (FUS+PLH+).

 

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  		Table 4. Response of three alfalfa (Medicago sativa L.) populations (FG1-1, FG2-1, LegenDairy) in Exp. 2 to four treatments: not inoculated, not infested (Control); inoculated with Fusarium (FUS+); infested with potato leafhoppers (PLH+); and Fusarium inoculated plus potato leafhopper infested (FUS+PLH+).

 
Crown plus Root Fresh Weights and Crown Rot Ratings
In Exp. 1, the PLH+ and FUS+PLH+ treatments resulted in reduced crown plus root fresh weights in all populations compared with the control (Table 3). Fusarium inoculation alone (FUS+) did not reduce crown plus root fresh weights in either of the Fusarium crown rot resistant populations (FG1-1 and FG2-1). In contrast, the Fusarium crown rot susceptible population (LegenDairy) had significantly lower crown plus root fresh weights than the control in Exp. 1. In Exp. 2, the only reduction in crown plus root fresh weights was observed in FG2-1 in the FUS+PLH+ treatment (Table 4). Neither FUS+ nor PLH+ treatments reduced crown plus root fresh weights in any population in Exp. 2.

The FG1-1 population showed significant differences in crown rot rating only between the FUS+PLH+ and the PLH+ treatments in Exp. 1 (Table 3). In FG2-1, treatments involving potato leafhopper infestation (FUS+PLH+ and PLH+) had lower crown rot ratings than the FUS+ and control treatments (Tables 3 and 4). In LegenDairy, crown rot ratings were highest in the FUS+PLH+ treatments in both experiments. In both Exp. 1 and 2, there were examples of comparable crown rot rating scores between Fusarium-inoculated treatments and uninoculated treatments by Week 35 (Tables 3 and 4). Low levels of F. oxysporum contamination were present in the uninoculated treatments by the termination of the experiment (Table 5), which was anticipated in experiments maintained for this duration despite precautions taken to minimize contamination. While it is possible that contamination accounted in some part for the elevated crown rot ratings in the uninoculated treatments, it is also possible that crown rot in these treatments was the result of low-grade necrotizing organisms colonizing tissue compromised by mechanical injury from harvesting or potato leafhopper infestations.


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  		Table 5. Percentage of alfalfa (Medicago sativa L.) crown and taproot segments colonized by Fusarium oxysporum in four treatments: not inoculated, not infested (Control); inoculated with Fusarium (FUS+); infested with potato leafhoppers (PLH+); and Fusarium inoculated plus potato leafhopper infested (FUS+PLH+).

 
Crown Symmetry Ratings and Presence of Secondary Crowns
There was a Fusarium main effect for crown symmetry in both experiments (Tables 1 and 2). Treatments with Fusarium were 0.2 (Exp. 1) and 0.3 (Exp. 2) units higher (less symmetric) in average crown symmetry rating than treatments without Fusarium (Tables 3 and 4). There was a potato leafhopper x population interaction effect for crown symmetry in both experiments (Tables 1 and 2). No loss of crown symmetry due to potato leafhopper was observed in FG1-1 in either experiment (Tables 3 and 4). In contrast, treatments with potato leafhopper infestation usually reduced crown symmetry in FG2-1 and LegenDairy, especially when combined with Fusarium inoculation (Tables 3 and 4).

There were no treatment effects on the percentage of survivors with secondary crowns in population FG1-1 in either experiment (Tables 3 and 4). For population FG2-1, there were no differences in percentage of survivors with secondary crowns at 35 wk in Exp. 1; however, in Exp. 2, the FUS+ and FUS+PLH+ treatments were lower than the control. In LegenDairy, all inoculation and infestation combinations resulted in decreased survivors with secondary crowns compared with the control in Exp. 1, while the FUS+PLH+ and PLH+ treatments reduced the number of survivors with secondary crowns in Exp. 2.

Yellowing Rating and Number of Potato Leafhoppers
Significant potato leafhopper x population interactions were observed for potato leafhopper yellowing and number of potato leafhoppers recovered from plants in Exp. 1 (Table 1). In the FG2-1 and LegenDairy populations, all treatments with potato leafhopper infestation had higher potato leafhopper yellowing ratings (3.7–3.9) than in FG1-1 (1.7). The ranking of populations for live potato leafhoppers recovered in infested treatments were: FG1-1 (39 leafhoppers pot–1) < LegenDairy (64 leafhoppers pot–1) < FG2-1 (98 leafhoppers pot–1). While the two potato leafhopper susceptible populations, FG2-1 and LegenDairy (lacking glandular hairs), had similar potato leafhopper yellowing ratings (3.7 vs. 3.9, respectively), the average number of potato leafhoppers present after 3 wk of infestation was significantly higher in FG2-1 than in LegenDairy in Exp. 1. There were no treatment differences in potato leafhopper numbers recovered in Exp. 2 with the exception of population FG2-1 (Table 4). In FG2-1, more potato leafhoppers were associated with the PLH+ treatment than in combination with Fusarium inoculation (FUS+PLH+), although no difference in potato leafhopper yellowing ratings was found between those two treatments. The potato leafhopper yellowing ratings were lower in the FG1-1 population, with the PLH+ treatment having lower ratings than in combination with Fusarium.

Colonization of Crown and Root Tissue by Fusarium oxysporum
Across both experiments and all populations, samples from treatments inoculated with F. oxysporum showed 56 to 98% colonization of crown and root tissue segments by F. oxysporum (Table 5). A relatively small portion of crowns and roots showed F. oxysporum contamination in the uninoculated treatments (Control, PLH+), with colonization ranging from 0 to 26%. No alfalfa-specific pathogens were observed from culturing crown and root tissues that might have been additional causal agents of crown rot.


   DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The difference in design between Exp. 1 and 2 was meant to simulate the differences that might occur in a typical alfalfa field situation in the U.S. Midwest. In Exp. 1, potato leafhopper infestation occurred before Fusarium inoculation, which corresponds to a spring-seeded alfalfa stand situation with potato leafhopper infestation occurring before the first harvest and Fusarium inoculation occurring at the first harvest. In Exp. 2, however, Fusarium inoculation occurred at the first harvest followed by potato leafhopper infestation on the regrowth at 2 wk, simulating the migration of potato leafhoppers into a field after the first harvest in a spring-seeded stand. Either situation is likely to occur in the north-central USA in newly seeded alfalfa stands in a typical year. While these two experiments were not conducted in a way that would allow direct comparisons between leafhopper infestation and Fusarium inoculation regimes (Exp. 1 compared with Exp. 2), one generalization that can be made is that, overall, the patterns of plant loss were similar when potato leafhoppers were introduced before Fusarium inoculation (Exp. 1) and when they were introduced onto alfalfa regrowth following the Fusarium inoculation (Exp. 2).

The results of Exp. 1 indicate that both Fusarium infection and potato leafhopper infestation reduce plant numbers with time, with most plant loss occurring in the brief period (two regrowth cycles) following Fusarium inoculation or potato leafhopper infestation. It is important to note that Exp. 1 was conducted in the greenhouse under the regime of a single Fusarium inoculation and a single potato leafhopper infestation. Under typical field conditions, alfalfa may be subjected to continuous, although potentially less thorough, Fusarium inoculations brought about by normal harvesting methods, and multiple infestations by potato leafhopper, although potato leafhopper populations are unlikely to reach the numbers in the field that were attained in the greenhouse cages. While most plant loss seemed to occur between the first and third harvests in both experiments in the absence of any continued Fusarium inoculations or potato leafhopper infestations, multiple cycles of inoculation and infestation may continue to cause plant loss in a field situation. Plant loss was associated with Fusarium inoculation in both experiments and indicated that Fusarium infection may affect persistence in the field.

Overall, the visual effects of potato leafhopper damage were very distinct. The populations lacking glandular hairs (FG2-1 and LegenDairy) showed more stunting, yellowing, and necrosis than the glandular-haired population (FG1-1) in both experiments. Early post-Fusarium inoculation symptoms were also observed in both experiments. Three days after inoculation, stubble to which Fusarium conidia had been applied showed discoloration and necrosis starting at the cut end of the stubble and progressing toward the crown, while the uninoculated controls showed healthy stubble, healing cleanly without discoloration or necrosis. Fusarium-inoculated plants were also noted to be visibly stunted compared with the uninoculated plants. There were no visual differences in symptoms among populations in the Fusarium-inoculated plants. In the regrowth cycle after inoculation, and throughout the experiment, there were no conspicuous foliar symptoms of Fusarium inoculation. It is also important to mention there were no symptoms observed that would be consistent with Fusarium wilt, causal agent Fusarium oxysporum Schlect. f. sp. medicaginis (Weimer) Snyd. et Hans., such as discoloration of the vascular tissue and wilting stems.

Previous studies have suggested that yield is not affected by Fusarium infection alone (Pegg and Parry, 1983; Gossen et al., 1994; Cormack, 1942; Hawn et al., 1981; Gossen, 1998), and results from both of the present experiments agree with these previous findings. As Fusarium crown rot is a disease that progresses slowly, it is possible that under field conditions reductions in yield would be minimal as long as no additional stresses are imposed. In LegenDairy, however, the combined stress of Fusarium infection and potato leafhopper feeding resulted in dry matter yield reductions of 72% in Exp. 1 and 76% in Exp. 2, suggesting that when a cultivar is susceptible to both stresses, substantial yield reductions may occur.

The colonization data indicate that infection by Fusarium may readily take place through the stubble generated at each harvest, as suggested by previous investigators (Wilcoxson et al., 1977; Richard et al., 1980; Turner and Van Alfen, 1983). Isolations from sampled root segments showed that colonization of roots by the Fusarium oxysporum isolate used for inoculation was more extensive at the end of 35 wk in Exp. 2 than in Exp. 1 (Table 5), indicating that the time at which potato leafhopper infestation occurs may be important in the interaction of the two stresses. The greater contamination by F. oxysporum in the LegenDairy control treatment (Table 5) may account for some of the reduction seen in plant numbers and yield for that cultivar in both experiments. While any stress to alfalfa has been implicated as predisposing plants to Fusarium infection and increasing crown rot severity, colonization by Fusarium oxysporum was more extensive when potato leafhoppers were applied after Fusarium inoculation (Exp. 2), compared with potato leafhopper infestation before Fusarium inoculation (Exp. 1). It is possible that the stunted growth brought about by potato leafhopper infestation resulted in fewer sites for potential infection or less tissue susceptible to Fusarium infection because of the reduced number of stems, less stubble, or a reduced rate of growth. This is not to suggest that potato leafhopper injury does not predispose alfalfa to infection by Fusarium, but there were relatively minor differences in all populations between the FUS+PLH+ treatments and the FUS+ treatments in the percentage of colonized roots at 35 wk in the two experiments.

The reduction in average crown plus root fresh weight per plant in the Fusarium-inoculated treatments and potato leafhopper infested treatments seen in Exp. 1 and the reduction in the FUS+PLH+ treatment in the potato leafhopper susceptible population FG2-1 in Exp. 2 is important in two ways. First, the crown serves as the region from which all regrowth is initiated. Second, it is generally accepted that taproot health plays a major role in alfalfa survival during winter. Hence, reduced crown plus root fresh weights resulting from Fusarium inoculation, potato leafhopper infestation, or both in combination, may impair the plant's ability to withstand winter conditions or reduce the initial vigor of the plants in the following season.

One possible explanation for the higher number of potato leafhoppers in the FG2-1 population in both experiments is simply that the inherent physiological characteristics or growth habit of the FG2-1 population supported higher numbers of potato leafhoppers than the LegenDairy population. In Exp. 1, the similarity in potato leafhopper yellowing ratings between FG2-1 and LegenDairy may suggest that a maximum level of damage was attained in both populations before the 5-wk potato leafhopper recovery event, regardless of any subsequent increase in potato leafhopper numbers. It is important to recognize that the population buildup of potato leafhoppers in Exp. 1 was very intensive and may not represent an average field situation. The numbers of potato leafhoppers recovered from FG2-1 and LegenDairy would have exceeded standard action thresholds in the field (Cuperus et al., 1983; Lefko et al., 2000; DeGooyer et al., 1998). Based on visual damage to the plants, it is likely that in the field, potato leafhoppers would have emigrated out of the alfalfa stand and the population would never reach a level comparable to those observed under cages in the greenhouse.

It should be noted that in neither experiment were leafhoppers present on plants at the time of Fusarium inoculation nor were potato leafhopper feeding or oviposition wounds present that could have provided entry points for Fusarium at the time of inoculation. In Exp. 1, potato leafhoppers were removed by vacuuming all potato leafhoppers from the foliage before making the first harvest. Also, as plant stems were harvested, all feeding or oviposition wounds were removed before inoculation. In Exp. 2, 10-wk-old plants were harvested and simultaneously inoculated with Fusarium 2 wk before potato leafhopper infestation. In both experiments, direct exposure between Fusarium and potato leafhoppers was minimal, suggesting that any significant interactions observed were probably the result of separate stresses imposed on the host. One noteworthy exception in the similarities of plant loss patterns between the two experiments occurred with FG2-1, the Fusarium crown rot resistant, potato leafhopper susceptible population. When potato leafhopper were introduced before Fusarium inoculation, the FUS+PLH+ treatment showed a virtually identical pattern of plant loss to that of PLH+ alone. In Exp. 2 when potato leafhoppers were introduced onto regrowth following harvest and inoculation, however, the FUS+PLH+ treatment showed substantially increased mortality over the PLH+ alone treatment, suggesting that potato leafhopper may interfere with host resistance mechanisms if potato leafhopper infestation occurs after Fusarium infection and initial colonization have taken place. What is more likely, however, is that there was a reduced amount of regrowth in the FUS+ treatments, thus resulting in a greater potato leafhopper stress (potato leafhopper per unit stem tissue) than on plants without Fusarium infection.

Based on the plant survival data and the Fusarium colonization data, there is evidence to suggest that Fusarium oxysporum may extensively colonize the crown and taproot of an alfalfa plant, with relatively minor effects until an additional stress is placed on the plant. The time period that the experiments were conducted was intended to simulate a 2-yr field interval. It is likely that the effects of crown rot, especially on yield components, may not become apparent until later in the life of the stand. Additionally, the experiments were conducted in the greenhouse in the absence of any winter stress. It is also possible that plant loss associated with Fusarium crown rot would be greater in a field situation where additional normal seasonal stresses are imposed. The general lack of any interaction between Fusarium infection and potato leafhopper injury suggests that the relationship is additive and largely dependent on the inherent characteristics of the individual alfalfa populations. Regrowth potential, both inherent ability and time of recovery, is a factor that seems to play an important role in persistence and yield in alfalfa populations exposed to the combined stresses of Fusarium infection and potato leafhopper infestation. Although evidence of an interaction between Fusarium and potato leafhopper was absent in these studies, these two stressors are nonetheless significant factors in stand and yield persistence of alfalfa. Dual resistance to both organisms was observed to enhance plant survival by 66 to 85% and increase yield 66 to 78% over a population susceptible to both organisms. Minimizing potato leafhopper injury, either through chemical control or the use of glandular-haired cultivars, may provide a benefit above simply protecting yields in that it also may lead to improved longevity in a production alfalfa stand.


   ACKNOWLEDGMENTS
 
We wish to thank Brian Kelly, Chris Boggs, Gretchen Sutton, Rebecca Lyon, Harold Brown, and Judy Smith for technical assistance and Sharie Fitzpatrick, Forage Genetics, for providing the three alfalfa populations.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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Received for publication October 18, 2006.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 





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