© 2008 Plant Management Network.
Foliar Symptom Expression in Association with Early Infection and Xylem Colonization by Fusarium virguliforme (formerly F. solani f. sp. glycines), the Causal Agent of Soybean Sudden Death Syndrome
Shrishail S. Navi and X. B. Yang, Department of Plant Pathology, 351 Bessey Hall, College of Agriculture and Life Sciences, Iowa State University, Ames 50011
Navi, S. S., and Yang, X. B. 2008. Foliar symptom expression in association with early infection and xylem colonization by Fusarium virguliforme (formerly F. solani f. sp. glycines), the causal agent of soybean sudden death syndrome. Online. Plant Health Progress doi:10.1094/PHP-2008-0222-01-RS.
Soybean sudden death syndrome (SDS), caused by Fusarium virguliforme (FV), is a root disease that results in severe foliar symptoms during the reproductive stage. In a regular growing season, an epidemic of SDS is highly correlated with the planting date and the disease tends to be more severe in earlier planted soybeans. Occurrence of infection early in the season is likely to result in colonization in the xylem and phloem tissues, a process essential for foliar symptom expression because xylem tissues are upward pathways in soybean plants. To demonstrate the biology of this infection, we used an effective and quantifiable seedling inoculation technique in which germinated seeds in a Petri dish were spray-inoculated with conidial suspension before being transplanted. Plants that had foliar symptoms showed both external and internal discolored taproots and basal stems, while plants with no foliar symptoms had only superficial discoloration. Microtome sectioning of taproots of plants that had foliar symptoms revealed the presence of fungal structures in both xylem and phloem tissues, while plants that had no foliar symptoms revealed fungal structures only in phloem tissue. A scanning electron microscope study showed a higher penetration frequency of FV near the root-cap zone where few or no root hairs of the radicle were found. These results indicate that fungal penetration into the xylem tissue plays a role in foliar symptom expression.
For nearly three decades, sudden death syndrome (SDS), caused by the soil-borne root pathogen Fusarium virguliforme O’Donnell et T. Aoki (2), formerly F. solani f. sp. glycines, has been recognized as a major disease of the soybean [Glycine max (L.) Merr.] in the southern United States. This disease was first reported in Arkansas in 1971 followed by Tennessee, Missouri, Mississippi, Illinois, Kentucky, Kansas, Indiana, Iowa, and more recently in Nebraska. In 1993, an SDS epidemic (46% of the soybean fields) was found in east-central Illinois (5) and there were several outbreaks in Iowa since then with fields having nearly 100% rate of infection (Fig. 1). Other countries that have reported SDS are Brazil (11), Argentina (16), and Canada (1).
In the past 10 years, SDS has become more prevalent and more severe in the northern soybean production areas of the United States, causing substantial yield reductions (22). The yield suppression due to SDS in the United States during 2002 was valued at $157.4 million (21). In a 1996 risk assessment study (17), Scherm and Yang predicted that the disease could become major production problem to north-central region of the United States although it was then considered a southern disease. The expansion of this disease has led to an increased concern among soybean producers in the north-central region. Yield losses due to SDS in the top 10 soybean producing countries range from 2 × 10³ metric tons in Canada to 9 × 105 metric tons in the United States (22), ranked as the top two fungal diseases.
Studies have shown that early soybean planting has been associated with increased severity of SDS (7,20); a cooler soil temperature (22 to 24°C) increases root infection by F. virguliforme in early-planted soybeans (17). Thus, delayed planting has been recommended as a management tactic for reducing SDS risk in the north-central production region (24). Although infections may occur early in the season, SDS causes characteristic foliar symptoms that are generally observed toward the flowering or pod-development stages as chlorotic, mottling, or mosaic on the upper leaves. Within a few days of symptom expression, chlorotic blotches that develop on leaves rapidly become necrotic and coalesce to form interveinal necrotic streaks (15,17,18,19).
Since root infection occurs prior to foliar symptoms expression, understanding root infection and colonization is important in relating above-ground symptoms expression to root colonization. There are reports that area under SDS fungus population curve was significantly correlated with the incidence of colonized roots and root colonization rate (10). The relationship between foliar symptoms expression and root colonization, as well as how root colonization develops in soybean cultivars was unknown.
Characteristic root symptoms observed in this study are discolored taproots and basal stems. These symptoms are the effects of F. virguliforme colonization in soybean phloem and xylem tissues. Knowing the location of infection of this pathogen and the time of occurrence of foliar symptoms, we understood to have severe SDS symptoms during the soybean reproductive stages; F. virguliforme needs to colonize in vascular tissue, which provides a pathway for upward movement of phytotoxin. Infection at the early seedling stage allows the pathogen to be effectively established in the xylem system, leading to foliar SDS symptom expression (23). Recently, preliminary reports of Yang and Navi (23) on this aspect were supported by Gao et al. (4) using real-time quantitative polymerase chain reaction. Infection later in the growth stage would lead to superficial colonization on the taproot outside the xylem tissue, with no effective upward movement of the toxin. Consequently, there would be no or much fewer foliar symptoms. Therefore, the objective of our study was to understand the process of infection and colonization by F. virguliforme in vascular tissue leading to discoloration and subsequent foliar symptom expression in basal stems and taproots.
A set of 24 soybean differentials (Table 1) were selected in 2003 for studies on F. virguliforme infection biology in the greenhouse experiments. These differentials were provided by Dr. M. E. Schmidt, Department of Plant, Soil and Agricultural Systems, Center of Excellence for Soybean Research, Teaching and Outreach, College of Agricultural Sciences, Southern Illinois University, Carbondale, IL 62901.
Table 1. Mean percent incidence, percent severity, and severity index of 24 differentials tested for resistance to soybean sudden death syndrome using dip-inoculation screening technique.
x Treatment means with the same letter are not significantly different.
y Severity index = number of SDS symptomatic plants × percent severity / total number of plants.
Inoculum Preparation and Inoculation
A single-spore isolate of F. virguliforme of the Mont-1 isolate from Illinois (putative virulent) was cultured on 9-cm-diameter disposable Petri dishes containing potato dextrose agar at one-third strength with 0.1 g streptomycin sulphate per liter. The isolate was incubated at 22 ± 1°C under a continuous photoperiod for one month. When the isolate was fully grown on the culture plate, 10 ml distilled sterile water (DSW) was transferred under aseptic conditions to release the fungal tissue, which was then stirred using a sterilized camel #8 brush, followed by a second 10-ml DSW wash. The resultant conidial suspension was transferred to a 100-ml sterilized graduated conical flask. The conidial population density was adjusted to 1 × 105/ml using a hemocytometer. The suspension was transferred to a 200-ml Nalgene aerosol spray bottle for inoculation.
For experiments on the mechanism of discoloration, seeds of the 24 differentials were germinated on moist paper towels and were transferred onto the lower lid of sterilized Petri dishes with 10 to 15 seeds per plate. These seeds were then spray-inoculated with a conidial suspension of F. virguliforme until the spore suspension dripped (Fig. 2). Thirty minutes post-inoculation, the excess spore suspension was decanted from the plates using a disposable sterile pipette, except from the vicinity of radicles. Untreated controls were maintained by inoculating germinated seeds with DSW. The plates were placed in the laminar flow approximately 2 h before they were transplanted in pre-irrigated Sweetheart plastic cups 13 cm high × 8 cm in diameter, drilled with seven 3-mm holes at the base (Sweetheart Cup Company, Chicago, IL).
Transplanting, Incubation, and Evaluation
Sweetheart plastic cups were filled to a depth of approximately 8 to 9 cm with a
mixture of sterilized soil and sand (2:1 ratio), with a further 3 to 4 cm depth
mixture of peat mix and soil (1:1 ratio) on top, leaving an approximate 2 to 3 cm
gap from the top rim of the cups. The soil was from a field cropped to soybean
that had no history of SDS and had the following characteristics: clay, 18%;
sand, 53%; pH, 6.6; and organic matter, 2.5% and the pH of sand was 5.8. Twelve
inoculated and four uninoculated germinated seeds from each of the 24 genotypes
were transplanted separately in pre-irrigated cups at four seeds per cup. The
cups were placed at random in Sterilite 30.28 liter clear view storage plastic
tubs, 16.18 cm high × 41.28 cm wide × 59.69 cm
long. Beginning at 24 h post-transplant
and continuing throughout the rest of the study, moisture in the cups was
maintained by providing about 5 cm water in the tubs. Plants were incubated on
greenhouse benches at 20 to 22°C under 400 watts light with a 16 h photoperiod for
a month. This experiment was carried out in a completely randomized block design
and was repeated once. Plants were evaluated for percent incidence, percent
severity, and severity of SDS once at 21 days and again at 30 days post-transplant.
The severity index was determined as follows:
Colonization by F. virguliforme in Phloem and Xylem Tissues
Seeds of three soybean genotypes (Cordell, Pharaoh, and Spencer) were germinated, inoculated, and transplanted separately in five cups for each genotype at four seeds per cup. Inoculum preparation, inoculation, and transplanting details were the same as described above. Controls were maintained by inoculation with DSW. Thirty days after transplanting, a set of four plants from each of the three genotypes that had (i) both foliar symptoms and external discoloration of basal stem and taproots; (ii) no foliar symptoms but external discoloration of basal stem and taproots; (iii) no foliar symptoms and no external discoloration of basal stem and taproots (uninfected healthy); and (iv) uninoculated healthy plants were sampled and stored in zip-lock plastic bags. The plants were thoroughly washed in running tap water and processed for microtome sectioning. In microtome sectioning the samples were fixed with 3% glutaraldehyde (w/v) and 2% paraformaldehyde (w/v) in 0.1 M cacodylate buffer, pH 7.2 for 48 h at 4°C. Samples were rinsed 3 times in 0.1 M cacodylate buffer and then post-fixed in 1% osmium tetroxide in 0.1 M cacodylate buffer for 1 h (room temperature). The samples were rinsed in deionized distilled water and dehydrated in a graded ethanol series, cleared with ultra-pure acetone, infiltrated and embedded using Spurr’s epoxy resin (Electron Microscopy Sciences, Ft. Washington, PA). Resin blocks were polymerized for 48 h at 65°C. Thick sections were made using a Reichert Ultracut Ultramicrotome (Leeds Precision Instruments, Minneapolis, MN). Images were scanned with a digital imaging system for computer enhancement to observe colonization of the fungus in phloem and xylem tissues.
Germination and Penetration Frequency of F. virguliforme
An experiment using two soybean genotypes, Ripley and P3981, was conducted under a controlled environment during 2004. One hundred and twenty seeds were germinated in a 9-cm humid-chamber Petri dish for a 24-h photoperiod at 22°C. Forty germinated seeds of each of the three radicle lengths (1 to 1.5, 2.5 to 3.2, and 4 to 4.3 cm) of Ripley and 0.6 to 1, 1.5 to 1.8, and 2 to 2.3 cm of P3981 were separately transferred into disposable Petri dishes and spray-inoculated with conidial suspension of F. virguliforme (1 × 05 conidia/ml) until the radicles were immersed in the suspension. The inoculated and uninoculated controls were incubated at 22°C for 1, 6, 12, and 24 h under a continuous photoperiod. After the incubation, radicles and cotyledons were separated at the intersection; the radicles were fixed with 3% glutaraldehyde, 2% paraformaldehyde in 0.1 M cacodylate buffer and dehydrated through a graded alcohol series. The specimens were dried using a critical-point dryer (Denton DCP II critical point dryer, Denton Vacuum LLC, Moorestown, NJ), mounted, and coated using a Denton Sputter coater (Denton Vacuum LLC) with a 60/40 palladium gold alloy target and viewed using a JEOL 5800LV scanning electron microscope. Images were scanned on a digital imaging system by computer enhancement to measure germination and penetration frequency of F. virguliforme.
ANOVA analysis for incidence, severity, severity index, mean proportion of basal stem and taproot external and internal discoloration, and their mean proportion of discoloration length (cm) were made following GLM procedure in SAS (SAS v. 9.1, SAS Institute Inc., Cary, NC).
Appearance of Symptoms
Foliar symptoms of soybean SDS were observed three weeks after transplanting. The characteristic foliar symptoms observed were interveinal chlorosis, interveinal necrosis, puckering, and mottling of leaves (Fig. 3). Plants that expressed foliar SDS symptoms had both external and internal discoloration of basal stems and taproots (Fig. 4). Plants with superficial root discoloration of the cortex tissue did not show foliar symptoms. Superficial discoloration of basal stems and taproots was an indication of superficial infection by the fungus, while the internal discoloration provided apparent indication of infection by the fungus and subsequent production of foliar symptoms. Soybean plants that had internal discoloration of taproot and the basal stem were followed with foliar symptoms expression with an exception of plants in Hutcheson and P3981 (Table 2) and P3981 in which a small proportion of plants had internal discoloration without foliar symptoms (Table 3).
Table 2. Mean proportion (percent frequency) of basal stem and taproots measured for discoloration inoculated with Fusarium virguliforme.
* Means with the same letter are not significantly different.
Table 3. Mean proportion of discoloration length (cm) of basal stem and taproots of plants that were inoculated with Fusarium virguliforme.
* Means with the same letter are not significantly different.
Incidence, Severity, and Severity Index
To determine incidence and severity, only the genotypes that had foliar SDS symptoms (Fig. 4) were considered. Mean incidence among 24 genotypes varied from 0% to 67%, severity from 0% to 75%, and the resulting severity index from 0 to 67 (Table 1). Genotypes with 0 to 10% incidence were Ripley, A 5560, Essex, and Manokin, and genotypes with ≥ 50% incidences were Cordell, Spencer, Pharaoh, and Spencer. The genotypes with a severity index of 0 to 10 were Ripley, Manokin, LS 90-1920, Essex, Egyptian, and A 5560; with 12 to 25 were A 4715, A 5403, Calland, CM 497, Forrest, Jack, and Pella 86; and there were 11 genotypes with the severity index of > 25 (Table 1).
Effects of Infection on Discoloration of Basal Stems and Taproots of Soybeans
In plants that had foliar SDS symptoms, 8.33% of plants of four genotypes (A 4715, A 5560, Essex, and Manokin) and 11 to 25% of plants of 11 genotypes (A 5403, Calland, CM 497, Douglas, Egyptian, Forrest, Jack, LS 90-1920, Pella 86, Spencer, and V 82-2191) showed both external and internal basal stem and taproot discoloration (Table 2). Of the 24 genotypes tested, only Hutcheson with 25% of plants and P3981 with 8.33% of plants showed internal basal stem and taproot discoloration with no foliar SDS symptoms. However, the other 22 genotypes did have foliar SDS symptoms with internal basal stem and taproot discoloration. Therefore, it appears that internal discoloration of basal stems and taproots may be necessary for foliar symptom expression.
Generally, the external discoloration length in basal stems and taproots was higher (0.55 to 7 cm) in plants that had no foliar SDS symptoms compared with plants that had foliar symptoms (0 to 4 cm) (Table 3). However, mean proportion of internal discoloration length of basal stems and taproots for plants that had foliar SDS symptoms varied from 0 cm in Ripley to 3.43 cm in Pharaoh. Interestingly, for reasons not known to us, of the 24 genotypes tested, only plants of P3981 with no foliar SDS symptoms showed 0.33 cm internal basal stem and taproot discoloration compared with plants of rest of the genotypes with no internal basal stem and taproot discoloration (Table 3).
Colonization in Phloem and Xylem Tissues of Soybean Taproots
Microtome studies of F. virguliforme colonization in Cordell, Pharaoh, and Spencer revealed the presence of hyphae in xylem and phloem tissues of taproots of the plants that had both foliar symptoms and external discoloration of the basal stem and taproots (Fig. 5). Hyphae were observed only in phloem tissue of taproots of plants that had no foliar symptoms but external discoloration of the basal stem and taproots (Fig. 6). However, no hyphae were observed in plants that had no foliar symptoms and no external discoloration of the basal stem and taproots or in uninoculated healthy plants. This indicates that there is an effective and ineffective colonization zone of this fungus in symptomatic and asymptomatic soybean plants, respectively (Fig. 7).
Germination and Penetration of F. virguliforme in Inoculated Soybean Seeds
Scanning electron microscopy of the germination and penetration of F. virguliforme in Ripley (Fig. 8A) and P3981 (Fig. 8B) shows (i) a lower conidial germination and penetration frequency in Ripley compared with P3981; (ii) a higher penetration frequency near the root-cap zone where few or no root hairs were observed in P3981; and (iii) penetration at the base of the root hairs in some sites but no distinct penetration in several sites (Table 4). We could not recognize the mode of entry of the germ tube into the xylem vessels from SEM, but we did observe formation of germ tubes, the appressorium, and the infection peg of the fungus on radicles.
Table 4. Mean percent germination and penetration of Fusarium virguliforme conidia on inoculated soybean seeds incubated for 6 hxy.
x Mean of 1000 sites of each radicle length.
y Germinated and penetrated conidia of F. virguliforme were counted from SEM images with magnification varying from 40× to 8000×.
Due to high processing costs, of the four incubation periods, only the 6 h incubation period samples (10 specimens from each of the radicle lengths) were processed and presented in Table 4. Of the 1000 sites of each radicle length observed, conidial germination varied from 86% to 92%, indicating that radicle length from 0.6 to 4.3 cm had no impact on conidial germination. However, there was a little variation in penetration percentage through root cap, root hairs, and base of root hair (Table 4).
Conclusions and Recommendations
From 3-year greenhouse/laboratory experiments, we concluded that colonization and penetration of F. virguliforme (formerly F. solani f. sp. glycines) in xylem tissues is critical for soybean plants to express SDS foliar symptoms. Although infection biology of the fungus is complex, our attempts seem to provide a better understanding of the mechanism of symptom expression. Together with follow-up work from another institute (4), this study helps understand the relationship between foliar symptoms expression and colonization by the fungus in root tissues (phloem and xylem) and, therefore, the importance of infection at an early seedling stage to foliar symptom expression.
Infected plants with foliar SDS symptoms had both external and internal discoloration in the taproots, while plants with superficial basal stem and taproot discoloration did not show foliar symptoms. In our investigation of the expression of SDS foliar symptoms in relation to the infection process, the results support our hypothesis of effective and ineffective zones of infection (Fig. 7). Therefore, inoculation methods providing consistent xylem infection are likely to be consistent in symptom expression. We also observed that the degree of discoloration (Table 2) indicates change in severity index values (Table 1) and proportion of discoloration length (Table 3), showing a positive relationship between severity index and degree of discoloration of the basal stem and taproots. Our results also support previous reports that demonstrated production of phytotoxins by F. virguliforme in SDS-infected soybean roots and their translocation to the leaves produce foliar symptoms (3,8,9).
Internal discoloration of basal stem and taproots in plants that express foliar SDS symptoms suggests that F. virguliforme colonizes vascular tissue by gaining entry through the radical’s root cap or the base of root hairs or epidermis (Figs. 8A and 8B) reach xylem tissue. This may eventually help translocation of toxin within the plant to produce foliar SDS symptoms. Secondly, in our investigation internal discoloration due to the infection was confined to the basal stem and taproots. This again complements earlier findings that the fungus can be isolated from the basal stems and roots but not from foliar parts (12,13). Further investigations are necessary to quantify what other fungal structures are in the basal stem and root zones. Our study presents similar evidence as has been reported in chickpea (6) for the requirement of internal stem and root discoloration for expression of foliar symptoms.
Microtome studies showed that F. virguliforme colonizes phloem and xylem tissues of soybean roots. The subsequent SDS symptoms expressed may be caused by translocation of phytotoxin within the plant. This study has contributed to the understanding of the relationship between planting date and SDS occurrence, and it provides an answer to the question of why early-planted soybeans are at a higher risk than those planted later, a question asked for years by soybean producers in the northern US soybean production regions. Scherm and Yang (17) showed that the SDS pathogen infects soybeans effectively in cool and wet soil. Soybeans planted early have slow germination and emergence, prolonging the contact period between the infective pathogen and soybeans. Since the infection can start as soon as a seed germinates, the earlier the planting, the longer the window of infection because of slow root growth and, consequently, the higher the risk of the disease (Yang and Navi, unpublished). Infection efficiency of F. virguliforme during a 36-h prolonged dipping in spore suspension showed similar levels of infection as a 1-h dipping (Navi and Yang, unpublished), indicating that an early infection in the radicle during seed germination might be sufficient for expression of symptoms. In addition, growth activities of both host and the pathogen were not suspended during the inoculation and incubation periods.
The SEM studies revealed germination and production of germ tubes, the appressorium, the infection peg on the radicles, and the mode of entry of the infection peg through the base of the root hairs and /or the root cap (Figs. 8A and 8B). However, we could not recognize the penetration of the appressorium and infection peg into the xylem or phloem vessels (Figs. 8A and 8B).
Our observations on possible avenues of infection in soybean plants are limited to what was shown in the microtome studies. Results of penetration frequency of F. virguliforme through the root cap or root hairs revealed the possibility of developing cultivars that resist avenues of these penetrations and of defining the optimal phytotoxin level to initiate the infection process. It may be essential to evaluate known resistant and known susceptible genotypes against different isolates.
We thank Dr Harry T. Horner, Tracey M. Pepper, and Randall L. Den Adel of the Bessey microscopy facility, Iowa State University (ISU), Ames, IA, for their technical support in processing samples for microtome and SEM during 2003-04. We also thank Dr T.C. Harrington at ISU for helpful discussions and Drs. Ram P. Thakur, ICRISAT, Patancheru, Andhra Pradesh 502 324, India, and Paul D. Esker at the University of Wisconsin-Madison.
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