Macrophomina phaseolina (charcoal rot of sweetpotato)

Taxonomy

Domain	Eukarya
Kingdom	Fungi
Phylum	Ascomycota
Subphylum	Pezizomycotina
Class	Dothideomycetes
Order	Botryosphaeriales
Family	Botryosphaeriaceae
Genus	Macrophomina

Scientific Name

Macrophomina phaseolina

Scientific Name Synonyms

Macrophomina phaseoli

Sclerotium bataticola

Botryodiplodia phaseoli

Tiarosporella phaseolina

Rhizoctonia bataticola

Macrophoma phaseolina

Common Name

charcoal rot

Author:Alan Henn, Mississippi State University

Pathogen

Macrophomina phaseolina is a Botryospaeriacea and can produce pycnidia in some media using defined light-dark cycles (Chidambaram and Mathur, 1975; Mihail, 1992; Goth and Ostazeski, 1965). Single-celled pycnidiospores are hyaline and 3:1 long:wide, about 4-30 x 5 – 10 μm. Pycnidia are not produced in sweetpotato tissue or other media referenced here.

Myceilia of the sclerotial stage are darkly pigmented with definite cell walls. They are somewhat sparse in media except in the areas where sclerotial production is under way. In older cultures sparse mycelia may become aerial, usually in pockets around the oldest sclerotia. Appearance of the mycelia varies somewhat. Three major types are encountered in Mississippi, but others may exist elsewhere. The pathogen produces a phytotoxin, botryodiplodin, that may facilitate infection (Ramezani et al. 2007).

The ubiquitous resting structures of the fungus are called both microsclerotia and sclerotia. They have somewhat diverse forms and sizes. Sclerotia of the same isolate generally measure larger when grown on plant tissue (about 100 – 1000 μm) than in culture (in water agar and Potato Dextrose Agar 50 – 300 μm). The shape can be ovid or spherical, but many are asymmetrical blocky. The sclerotia are produced more or less evenly within areas that are the same age, without dense patches, unless immediately surrounding early growth on plant tissue. When grown from plant tissue, a mycelial strand will leap from an older sclerotia to form a newer sclerotia, so single strand chains are not uncommon.

Infection of sweetpotato by Macrophomina phaseolina is frequently accompanied by Fusarium spp. Ten Fusarium species have been isolated from Mississippi sweetpotatoes in conjunction with Macrophomina phaseolina (Stokes et al. 2013c), of which six were pathogenic (Stokes et al. 2013b). Pathogenic Fusarium oxysporum and F. solani isolates were found by Stokes et al. (2013a, c) to be in 70% of the samples isolated from field grown tissue, but decreased to 27% in samples drawn from roots stored for 60 or 90 days.

The general presentation of the combination of pathogenic Fusarium species and Macrophomina phaseolina is a “wet” rot. Unless the front of the Fusarium wet rot is moving quite rapidly, some dark mycelia and sclerotia of Macrophomina phaseolina will be present in parts of the storage root.

Symptoms and Signs

Macrophomina phaseolina infection of sweetpotato roots may be asymptomatic or symptomatic (da Silva and Clark 2013, Stokes ???). If symptomatic, the disease usually expresses in storage (Ramsy, 1931, Lauritzen 1935, Ray and Edison 2005, Arancibia et al. 2013, da Silva and Clark, 2013) often on one end or the other of the sweetpotato storage root, although the stem end is most common (Ray and Edison, 2005, Davis, 2014, Henn, unpublished). Disease expression varies among the California, Louisiana, Mississippi, and North Carolina growing areas.

The leading edge of Macrophomina phaseolina infection in sweetpotato storage root tissue is somewhat firm and light-brown, tinged red in color (Figure ). The area behind the leading edge becomes darker as more sclerotia are formed (Figure ). The common name “charcoal rot” comes from many hundreds of black charcoal-colored sclerotia populating infected tissue. The density of sclerotia in a given age area are more or less uniform. In culture, three different morphological forms of growth of pathogenic Macrophomina phaseolina are present in Mississippi (Stokes et al., 2013). A description of these growth types is in the “Diagnostic Procedures” section.

Macrophomina phaseolina infection in stored sweetpotato roots can be symptomless (da Silva and Clark, 2014, Stokes, ???). Perhaps physical damage to the root or a conducive environment are needed to trigger disease symptom development in asymptomatic sweetpotatoes (da Silva and Clark, 2012).

If the disease expresses in storage, and environmental conditions are favorable for transpiration, infected root parts or roots will shrink and dry with the sweetpotato periderm (or skin) remaining intact (Clark et al. 2013) (Figure ). The disease may become restricted to part of the sweetpotato storage root so that one end may be desiccated and the other appear intact. The mummies can be found in storage and in field from harvest through spring planting (Figure).

Infection of sweetpotato by Macrophomina phaseolina is frequently accompanied by Fusarium spp. Ten Fusarium species have been isolated from Mississippi sweetpotatoes in conjunction with Macrophomina phaseolina (Stokes et al. 2013c), of which six were pathogenic (Stokes et al. 2013b). Pathogenic Fusarium oxysporum and F. solani isolates were found by Stokes et al. (2013a, c) to be in 70% of the samples isolated from field grown tissue, but decreased to 27% in samples drawn from roots stored for 60 and 90 days.

Disease expression varies among the California, Louisiana, Mississippi, and North Carolina growing areas. Macrophomina phaseolina disease of sweetpotato is prevalent and aggressive in Mississippi (Arancibia, et al. 2013, Burdine, 2008) where it has been called stem end rot or commonly called “end rot”. Symptoms present as either “wet” or “dry” (Figure 1 a-xx). End rot disease also occurs in Louisiana, but usually with a slight variation. The variation typically consists of small internal charcoal-colored spots of rot within the parenchyma, a symptom sometimes called “pocket rot”. There are few external symptoms. In North Carolina the expression changes to more of an internal lesion, accompanied by pocket rot-like symptoms. The disease is named internal necrosis. In California, Macrophomina phaseolina is an occasional problem on water stressed plants during high temperatures, and is seen on stems and as a dry stem end rot (Davis, personnel communication). The disease is not reported in New Jersey.

Geographic Distribution

Macrophomina phomina is a cosmopolitan pathogen found in many countries (Farr and Rossman, 2014) and at least 500 different hosts (Farr and Rossman 2014, Ramezani et al. 2007). It is a known pathogen of major agronomic crops.

Management

Disease management is not very advanced. There are three significant areas where management may be effective: 1). Slip production 2). Field production and 3). Storage.

Slip production

Work by Stokes et al. ( ) has shown that the sweetpotatoes used for slip production, and some slips produced by them harbor the pathogen. If pathogen growth in the slip production beds can be limited, health of the slips might be improved.

Increased cutting height of the slips would be beneficial, but cutting height is usually adjacent to the soil where growth from infected sweetpotatoes and from soil splashed propagules infect the base of the cut slips. Raising the cutting height will increase the amount of time for sufficient slip length and reduce the number of cuttings growers can obtain from a given bed. This approach will be difficult to encourage.

Fungicides or splash barriers such as mulches might be helpful. Labeled products change, but two currently labeled products include Botran, Quadris and many biologicals.

Field production

Reduce plant stress. Plant stress may encourage infection by the pathogen. The herbicides chloramben and 2,4-DB encourage root colonization by Macrophomina phaseolina of soybean (Canaday et al. 1986). Application of the ethylene precurser, Ethophon, to sweetpotato is related to restricted tip rot (two year combined correlations (r) of 0.54-0.78 (Arancibia et al. 2013)(Figure ), and sometimes can be related to Macrophomina phaseolina infection. Correlations between the number of sclerotia in the soil at harvest with the number of reniform nematodes at planting have been notable in some trials.

Informal grower trials showed that soaking crates full of cut slip bases with 3 pounds of RootShield/100 gallons water for about 45 minutes prior to transplant seemed to reduce pack out losses from the disease.

Fumigation trials using Telone II and Vapam in fields infested with reniform nematodes generally did increase different aspects of yield but showed a non-significant trend in reducing storage losses.

Losses are worse when sweetpotatoes are harvested after wet weather. If possible, a growing interval between heavy rains and harvest operations may be beneficial.

Storage

Most losses seem to occur to growers who do not cure harvested sweetpotatoes before storage at about 68 F. Good curing practices should reduce storage losses. Keep harvested sweetpotatoes away from heat sources.

Diagnostic procedures

Earlier workers thought Macrophomina phaseolina infection occurred at harvest, invading the sweetpotato through wounds created during the harvest operations (Lauritzen, 1935, Clark et al., 2013). Recent work has shown that Macrophomina phaseolina infection can occur 45 days after transplant in immature storage roots (Woolfolk, ????) and in small feeder roots as early as 10 days after transplant (Henn unpublished). Macrophomina phaseolina increases from under 2% of isolates made 45 days after transplanting to 6.5% of the isolates made from sweetpotatoes stored for 60 or 90 days (Stokes et al. 2013). Thus, diagnostics may be required for any plant part or for estimation of the sclerotia in soil

The optimal growth temperature for Macrophomina phaseolina in culture seems to be about 31° C (Barghava, 1965, Clark et al. 2013, Lauritzen 1935, Henn, unpublished). Most isolation procedures should be carried out in this temperature range.

There are at least three types of Macrophomina phaseolina growth forms in Mississippi. The most common is a light-gray myceilium that is more “fluffy” and profuse than the other Macrophomina types. The second produces sparser mycelia, is dark, and microsclerotia and mycelia will often color the media. The third produces sparse mycelia and does not seem to produce microsclerotia.

Isolation from soil Henn (unpublished) looked at various published methods for isolating Macrophomina phaseolina from soil, including methods from Mihail (1992), Campbell and Nelson (1986), and Cloud and Rupe (1991). The laters RB medium is in the Diagnosticians Cookbook (http://wiki.bugwood.org/RB_media_for_Macrophomina). The methods were compared using two soil types (silt loams vs. a heavy silt/light clay loam) from Mississippi sweetpotato fields. The RB medium and technique of Cloud and Rupe resulted in much higher recoveries (ca. 17x) and lower standard deviations (less than 10% of the mean) than did the Campbell and Nelson method.

Subsequent use in more soil types found that addition of other fungicides to the RB base could be helpful, depending on the specific soil and its problematic flora. Specifically, metalaxyl or tachigarn for suppression of oomycetes/pythium may be needed in some soils. Addition of thiophanate-methyl may assist in other soils. Counts seemed to be similar in soils tested with and without thiophanate-methyl, but excess growth of other fungi on non-treated plates make the conclusion questionable. The hybrid RB recipe appears at the end of this section.

Isolation from plant tissue Plant tissue from leaves, stems and roots work best if surface sterilized for 3 minutes in 0.525% sodium hypochlorite, rinsed three times in sterile water baths for 3 minutes each, and blotted dry on autoclaved filter paper. The surface sterilized pieces can be plated on potato dextrose agar (PDA), acidified PDA, corn meal agar and water agar.

For cleanness, water agar works well. Unlined, autoclaved index cards can be added to the media. The sclerotia will preferentially grow on these pieces of card stock. Growth will generally require 5 or more days. Sections of unlined card stock (ca. 1 cm x 0.5 cm or 1.5 cm x 1 cm) should be autoclaved and dried. As the 50 – 60° C media is poured, the card stock can be slipped into the media using sterilized tweezers.

Sweet potato root tissue ca. 1 x 1 x 1 cm can do ok with surface sterilization but the outside 5 mm of the tissue should be removed with a flamed scalpel prior to plating.

Humid box incubation works for sweetpotato storage root tissue, but may require as long as 10 – 16 days of incubation. Do not get the substrate too wet and check for humidity several times during incubation.

(Hybrid RB Macrophomina recipe for Macrophomina from soil and roots)

Resources and References

• Arancibia, R. A., J. L. Main and C. A. Clark. 2013. Sweetpotato tip rot incidence is increased by preharvest applications of ethephon and reduced by curing. HortTechnology 23(3): 288-293.
• Artschwager, E. F. and R. C. Starrett. 1931. Suberization and wound-periderm formation in sweetpotato and gladiolus as affected by temperature and relative humidity. Journal of Agricultural Research 43:353-364.
• Baird, R. E., P. A. Wadl, T. Allen, D. McNeill, X. Wang, J. K. Mouton, T. A. Rinehart, H. K. Abbas, T. Shier, R. N. Trigiano. 2010. Variability of United States isoloates of Macrophomina phaseolina based on simple sequence repeats and cros genus transferability to related genera within Botryosphaeriacae. Mycopathologica 170:169-180.
• Bhargava, S. N. Studies on charcoal rot of potatoes. Journal of Phytopathology 53(1):35-44.
• Campbell, C. Lee, and L. A. Nelson. 1986. Evaluation of an assay for quantifying populations of sclerotia of Macrophomina phaseolina from soil. Plant Disease 70:645-647.
• Canaday, C. H., Helsel, D. G., and T. D. Wyllie. 1986. Effects of herbicide-induced stress on root colonization of soybeans by Macrophomina phaseolina. Plant Disease 70:863-866.
• Chidambaram, P. and S. B. Mathur. 1975. Production of pycnidia by Macrophomina phaseolina. Transactions of the British Mycological Society 64:165-168.
• Clark, C. A., D. M. Ferrin, T. P. Smith, and G. J. Holmes, eds. 2013. Compendium of Sweetpotato Diseases, Pests and Disorders, 2nd ed. American Phtopathological Society, St. Paul, MN.
• Cloud, G. L., and J. C. Rupe. 1991. Comparison of three media for enumeration of sclerotia of Macrophomina phaseolina. Plant Dis. 75:771-772.
• Davis, R. M. 2014. Personal communication.
• Farr, D.F., & Rossman, A.Y. 2014. Fungal Databases, Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved July 16, 2014, from http://nt.ars-grin.gov/fungaldatabases/
• Goth, R. W. And S. A. Ostazeski. 1965. Sporulation of Macrophomina phaseoli on propylene oxide-sterilized leaf tissues. Phytopathology 55:1156.
• Lauritzen, J. I. 1935. Factors affecting infection and decay of sweetpotatoes by certain storage rot fungi. Journal of Agricultural Research 50: 285-334.
• Mihail, J. D. 1992. Macrophomina. In Methods for research on soilborne phytopathogenic Fungi. Singleton, L. L., J. D. Mihail, and C. M. Rush. APS Press. St. Paul, MN.
• Ramezani, M., W. T. Shier, H. K. Abbas, J. L. Tonos, R E. Baird and G. L. Sciumbato. 2007. Soybean charcoal rot disease fungus Macrophomina phaseolina in Mississippi produces the phytotoxin (-)-Botryodiplodin but no detectable phaseolinone. J. Natural Products 70:128-129.
• Stokes, C. E., et al. "Diversity, densities, and distribution of microbial communities in sweetpotato end/tip rot diseases." HortScience 47 (2012): S47-S48.
• Stokes, C.E., S.W. Woolfolk, R.A.Arancibia, and R.E.Baird. 2012. Diversity, densities, and distribution of microbial communities in sweetpotato tip/end rot diseases. Proceedings of the National Sweetpotato Growers Council Annual Meeting.
• Stokes, C. E., R. A. Arancibia and R. E. Baird. 2013a. Diversity and density indices of the pathogenic microbial community present in tip/end rot disease of sweetpotato. HortScience
• Stokes, C. E., R. A. Arancibia, and R. E. Baird. 2013b. Microbial disease complex of sweetpotato (Ipomoea batatas L. Lam.) tip/end rot. Phytopathology. Vol. 103. No. 6. 3340.
• Stokes, C.E., R.E. Baird, R.A. Arancibia, and T.W. Allen. 2013c. Diversity of fungal and bacterial communities in tip/end rot diseased sweetpotatoes. (Abstr.) Phytopathology 103 (Suppl. 1):S1.1. http://dx.doi.org/10.1094/PHYTO-103-5-S1.1
• MSA Citation - abstracts from 2013 have not been published in 'Inoculum' yet... sorry.
• YOUNG, P. A., 1949: Charcoal rot of plants in Eastern Texas. Texas Agric. Exp. Sta. Bull. 712, 1—33.

Acknowledgements

• National Institute of Food and Agriculture, U.S. Department of Agriculture, under Agreement No. 2011-41530-30708 as part of "Diagnostic Image Series Development for Supporting IPM in the Southern Region" (USDA-NIFA-RIPM-003351)