Evaluation of two methods to infect soybean with Macrophomina phaseolina (deuteromycota) under controlled environmental conditions.

ABSTRACT

To investigate plant resistance mechanisms or control techniques, a method for consistent infection of soybean by Macrophomina phaseolina must be used. The objective of this research was to evaluate the effectiveness of two methods to inoculate soybeans with M. phaseolina under controlled environmental conditions. A single isolate of M. phaseolina was introduced into soybean stems by puncturing a hanging drop from a prepared sclerotial suspension or by adding a cornmeal/sand culture mixture into potting soil prior to planting soybean in a greenhouse. Infection was determined visually by the presence of charcoal rot lesions on root tissue and by laboratory culture of root sections. Results showed that there was no difference in the incidence of M. phaseolina in soybean for either inoculation method used in this study. The cornmeal/sand method and the hanging drop method resulted in 58% and 45%, respectively. Application of a lime, peat, or starch based seed coating did not significantly (P=0.05) change the infection rate compared to no seed coating. Either method could be used for M. phaseolina infection of soybean under controlled environmental conditions. However, the ease of using the cornmeal/sand method compared to the hanging drop method makes it a preferable technique for soybean pathology research.

INTRODUCTION

Macrophomina phaseolina (Tassi) Goid. is a soilborne pathogen that causes charcoal rot of soybean. Soybean yield and seed quality are reduced when disease symptoms are severe. Host seeds are suspected of carrying M. phaseolina within the seed coat as ectophytic and/or endophytic sclerotia and hyphae. These serve as effective propagules for dissemination of the pathogen (Nyvall, 1999). Sclerotia remain viable for less than 8 weeks if exposed to continuously wet soils. Fungal growth and disease symptoms increase rapidly when ambient temperatures are between 28[degrees] and 35[degrees] C (Gray, 1989; Bowers and Russin, 1999). M. phaseolina can infect 100% of seedlings emerging in infested soil within 2-3 weeks following planting [Wyllie (1976) in Bowers and Russin (1999)]. Infection rate increases with higher soil temperatures and low soil moisture. Also, acidic (pH 4.3) and alkaline soils (pH 8.0) generally cause increased sclerotial production.

In the soil, sclerotia of M. phaseolina germinate on host plant root surfaces and produce multiple germ tubes (Gray, 1989). Root penetration usually occurs when appressoria form over anticlinal walls of epidermal cells, however, penetration can also occur through natural openings. Fungal hyphae will initially grow intercellularly and subsequently grow intracellularly through the xylem of the plant. Disease symptoms are more evident in drought-stressed plants due to disruption of already limited water and nutrient movement within the host plant. Most root damage, which later contributes to yield reduction, occurs early in the growing season. However, disease symptoms are not evident until the beginning of flowering when sclerotia begin to appear. Infected soybean plants will senesce prematurely; foliage will appear chlorotic and pods fail to fill completely (Bowers and Russin, 1999). To investigate plant resistance mechanisms or control techniques, a method for consistent infection of soybean by M. phaseolina must be used. The objective of this research was to evaluate the effectiveness of two methods to infect soybeans with M. phaseolina under greenhouse growing conditions.

MATERIALS AND METHODS

A single isolate of M. phaseolina (obtained from R. Baird, Dept. Entomology and Plant Pathology, Mississippi State University, Mississippi State, MS) was used in all experiments. The isolate was from cotton (Gossypium hirsutum L.) grown in Lee County, MS in 1999 and was cultured on potato dextrose agar (PDA) at 22[degrees] C ([+ or -] 2[degrees] C) under ambient conditions of 12 hour [day.sup.-1]. PDA was comprised of 39 g of agar [liter.sup.-1] of distilled water plus 0.25 ml Danitol[R]. In addition, stock solutions of 0.50 ml of chlortetracycline, and 2.5 ml of streptomycin were added to agar to prevent bacterial growth. Research was conducted at the Foil Plant Science Research Center (FPSRC) greenhouse complex located at Mississippi State University to determine a consistent method to introduce M. phaseolina into host soybean plants. The commercial soybean cultivar, Hutcheson, was selected for use throughout the experiment. This variety was chosen because it is a late maturing variety (maturity group V) adapted to environmental growth conditions occurring across the east coast and the southern United States. A peatmoss seed coating (PSC), lime seed coating (LSC), and starch seed coating (SSC) were prepared on the day of planting to evaluate their effects on infection of soybean seedlings by M. phaseolina. For clarity, seed coatings will refer to material applied to the seed, and seed treatment will refer to actual treatments with or without a seed coating. Peatmoss (Premier Horticulture Inc., Red Hill, PA) (pH 4.0) was passed through a 1 [mm.sup.2] gap wire mesh sieve and then coated onto inoculated seed via rotation in an Erlenmeyer flask. The same procedure was used to coat the seeds with pulverized dolomitic limestone (pH 8.5) (Southdown Inc., Easton, PA). The starch seed coating was made by adding 20 g of cornstarch (pH 5.3) to 300 ml distilled water, heating to a boil, and thoroughly cooling. This material was then coated onto the seed following application of the fungal suspension. Potting soil (Pioneer Southern, Lancaster, NY) (pH 7.0) was placed into 11 L plastic pots, and soybean seeds were planted into each pot at a depth of 2.5 cm. Each treatment consisted of 20 pots containing three plants each. Each pot was an experimental unit within the seed treatments. At 30 days post-emergence, M. phaseolina was introduced directly into seedlings. Using a modified technique described by Hanson (2000), a droplet from a suspension containing three sclerotia [ml.sup.-1] was applied to the seedling stem at 2 cm below the first cotyledonary node with a 26-gauge needle. The needle was punctured through the inoculum droplet and into the stele of the seedling. Each seedling was inoculated a second time on the opposite side of the stem, similar to the first inoculation. Controls were inoculated twice with sterile water. For the remaining experimental units, M. phaseolina was infested into the soil at 30 days post-emergence using a modified technique described by Baird et al. (1996). M. phaseolina was cultured in sterilized cornmeal/sand mixtures (CMS) in 1L Erlenmeyer flasks for 14 days at 22[degrees] C. Flasks were rotated each day for 5 min to establish uniform growth of M. phaseolina. Fifty ml of infested CMS were then mixed into the upper 4 cm of soil in each experimental unit. Experimental units included as controls were not infested.

At 90 days post-emergence, all plants were excised from pots and visual disease severity (VDS) represented as charcoal rot lesions on root tissue was determined using a modified disease index as described by Chakraborty and Purkayastha (1983). After disease symptoms were rated, five plants were randomly chosen from each inoculation method and seed treatment. Two 1 cm root tissue pieces from the upper portion of the tap root were excised and surface sterilized in 0.5 % NaOCl for 2-3 min, and plated on water agar (WA) containing chlorotetracycline and streptomycin. Plates were incubated at 22[degrees] C and examined every 2 days for 2 weeks or until signs of fungal growth were visible. Fungal isolates were taken from each active root tissue culture and transferred to PDA containing antibiotics and miticide and were allowed to grow for 7 days. M. phaseolina isolates from root tissue were identified using characteristics described by Rifai (1969) and Watanabe (1994).

The experimental design was completely randomized with 20 replications in a two-way factorial arrangement of treatments. The procedure general linear model (PROC GLM; SAS, 1994, SAS Institute, Inc., Cary, NC) was used to account for uneven data sets, and means were separated by Fisher's protected least significant difference (LSD).

RESULTS

There was no difference in the incidence of M. phaseolina in soybean for either inoculation method used in this study (Table 1). M. phaseolina was present for all seed treatments in which the soil was infested with cornmeal sand inoculum or when the hanging drop infestation method was used (Table 2). Within the cornmeal sand inoculation method, plants from all seed treatments, except the control, had isolation ratings for M. phaseolina that were not significantly different from the MP treatment (Table 2). The LSC and PSC isolation ratings for M. phaseolina from root tissue were not different from the untreated control. Within the hanging drop method, plants from all seed treatments had isolation ratings for M. phaseolina that were not significantly different from the MP treatment, except the untreated control. The LSC isolation ratings were not different from the untreated control.

Plants from the MP seed treatment had the greatest VDS ratings for both inoculation methods. VDS ratings for MP seed treatment were not different from the LSC treatment in the cornmeal/sand method (Table 3). Both PSC and SSC seed treatments resulted in lower VDS ratings regardless of the method used to introduce inoculum. VDS ratings were lowest for the untreated control in both introduction methods.

DISCUSSION

Willey (1976) reported a potential for 100% infection of soybean seedlings by M. phaseolina within 2-3 weeks following planting. Although this percentage was not reproduced in this study, an infection level of 78% was achieved for plants growing in a potting mixture infested with cornmeal/sand inoculum. The fluctuation of the soil environment in the greenhouse and pot conditions may have impacted the level of M. phaseolina infestation in our study, as earlier implicated by Willey (1976).

Inoculum prepared in cornmeal/sand did not result in greater isolation ratings for M. phaseolina from root tissue compared to isolations from plants inoculated by the hanging drop method. These results were similar to a previous study conducted by Hsi (1961) in which sorghum seeds were planted into soil naturally infested with M. phaseolina. In a previous study (Young, 1943), seedlings were artificially inoculated with the pathogen at 2 weeks after pollination using an infested toothpick. The artificial inoculation method from such study did not result in disease that was significantly different from disease developing in plants grown in naturally-infested soil.

The application of selected seed coatings reduced infection of soybean seedlings by M. phaseolina using either inoculation method. However, the reductions were not statistically different in this study. Disease development was also reduced for all seed treatment applications except one treatment. The numerical reduction in isolations of the fungus, therefore, has as yet unexplained implications in subsequent disease development. Seed coatings such as PSC and SSC should be avoided when conducting experimentation on M. phaseolina infection of soybean under greenhouse culture. Results of the current study argue for the use of untreated seed in such studies to maximize infection and disease development.

Although VDS provided a quick estimate of disease, it appeared to over estimate infection level but was consistent with the presence or absence of M. phaseolina. Under conditions of this study, the pathogenicity of the fungus could depend on factors independent of infection level.

CONCLUSIONS

The soilborne pathogen, M. phaseolina, is the causal organism of charcoal rot of soybean. This fungus causes external and internal damage to the host and often reduces soybean yield and seed quality. Infection by M. phaseolina did not differ whether inoculum was prepared in cornmeal/sand or introduced directly into the soybean seedling. Based on these results, future research which utilizes the cornmeal/sand method will benefit from the simplicity of inoculum preparation and the lack of damage to soybean seedlings during the infection process. The cornmeal sand method specifically eliminates puncture wounds to soybean seedlings which are a necessary element of the hanging drop method of inoculation. Use of VDS to quantify levels of M. phaseolina infection was not consistent with true isolation ratings but did provide a distinct evaluation of the level of disease development on soybean caused by this organism.

REFERENCES

Baird, R.E., D.E. Carling, and B.G. Mullinix. 1996. Characterization and comparison of isolates of Rhizoctonia solani AG-7 from Arkansas, Indiana, and Japan, and selectAG-4 isolates. Plant Dis. 80:1421-1424.

Bowers, G.R., and J.S. Russin. 1999. Soybean disease management. Pages 233-235 in L.G.

Heatherly, and H.F. Hodges eds. Soybean production in the midsouth. CRC Press LLC, Boca Raton, FL.

Chakraborty, U., and R.P. Purkayastha. 1983. Role of rhizobitoxine in protecting soybean root from Macrophomina phaseolina infection. Can. J. Microbiol. 30:285-289.

Gray, L.E. 1989. Charcoal Rot. Pages 34-38 in J.B. Sinclair, and P.A. Backman eds. Compendium of soybean diseases, 3rd Ed. APS Press, St. Paul, MN.

Hanson, L.E. 2000. Reduction of verticillium wilt symptoms in cotton following seed treatment with Trichoderma virens. J. Cotton Sci. 4:224-231.

Hsi, C.H. 1961. An effective technique for screening sorghum for resistance to charcoal rot. Phytopathology 51:340-341.

Nyvall, R.F. 1999. Diseases of soybean. Pages 634-636 in R.F. Nyvall ed. Field crop diseases, 3rd Ed. Iowa State University Press, Ames, IA.

Rifai, M.A. 1969. A revision of the genus Trichoderma. Pages 31-38 in M.A. Rifai ed. Mycological papers No. 116. Commonwealth Mycological Institute, Kew, Surrey, England.

SAS Institute, Inc. 1994. SAS user's guide: Statistics. SAS Institute, Inc., Cary, NC.

Watanabe, T. 1994. Morphology of soil fungi Deuteromycotina. Pages 264-382 in T. Watanabe ed. Pictorial atlas of soil and seed fungi morphologies of cultured fungi and key to species. CRC Press, Inc., Boca Raton, FL.

Willey, T.D. 1976. Macrophomina phaseolina--charcoal rot, Pages 482-484 in L.D. Hill ed. World Soybean Research, Proceedings of the World Soybean Research Conference, Interstate, Danville, IL.

Young, H.C. 1943. The toothpick method of inoculating corn for ear and stalk rots. (Abstr.) Phytopathology 33:16.

S. Bell Surrette (1), P.D. Meints (2), and L.E. Trevathan (3)

(1), Dept. of Biological Sciences, 124 Shoemaker Hall, University of Mississippi, University, MS. (2) Dept. of Plant and Soil Sciences, Box 9555, Mississippi State Univ., Mississippi State, MS. (3) Dept. of Entomology and Plant Pathology, Mississippi State University, Mississippi State, MS.

S. Bell Surrette Corresponding author

Table 1. Mean isolation rating (MIR) for the presence of Macrophomina
phaseolina in soybean root tissue taken 90 days post-emergence from
seedlings planted in a greenhouse and inoculated by two different
methods.

                         Mean Isolation Rating *
               Cornmeal sand method  Hanging drop method

M. phaseolina  0.58 a ([dagger])     0.45 a

* 1 = present in culture; 0 = not present in culture
([dagger]) Means followed by the same letter are not significantly
different according to Fisher's LSD (P = 0.05) test.

Table 2. Mean isolation rating (MIR) for the presence of Macrophomina
phaseolina in soybean root tissue taken 90 days post-emergence from soil
containing cornmeal/sand inoculum or from seedlings inoculated with a
hanging drop of sclerotial suspension.

                        Mean Isolation Rating *
Seed Treatment  Cornmeal sand method  Hanging drop method

MP              0.78 a ([dagger])     0.50 a
LSC             0.40 ab               0.29 ab
PSC             0.45 ab               0.72 a
SSC             0.75 a                0.60 a
C               0.13 b                0.00 b

* 1 = present in culture; 0 = not present in culture
([dagger]) Means followed by the same letter within a column are not
significantly different according to Fisher's LSD test (P = 0.05).
MP = M. phaseolina only, no seed coating; LSC = lime seed coating;
PSC = peat seed coating; SSC = starch seed coating; and, C = untreated
control.

Table 3. Visual disease severity (VDS) ratings for charcoal rot in
soybean root tissue taken 90 days post-emergence after growing in soil
containing cornmeal/sand inoculum of M. phaseolina or from seedlings
inoculated with a hanging drop of sclerotial suspension.

                     VDS Rating as Percent Infection
Seed treatment  Cornmeal sand method  Hanging drop method

MP              87 a ([dagger])       96 a
LSC             91 a                  55 b
PSC             44 c                  56 b
SSC             65 b                  45 b
C                3 d                   0 c

([dagger]) Means followed by the same letter within a column are not
significantly different according to Fisher's LSD test (P = 0.05)
MP = M. phaseolina only, no seed coating; LSC = lime seed coating;
PSC = peat seed coating; SSC = starch seed coating; and, C = untreated
control.