Variations in Pathogenicity of Three Different Forms of Rhizoctonia Inoculum and Assessment of Cultivar Resistance of Sugar Beet

Research Article

J Pathol & Microbiol. 2021; 3(1): 1019.

Variations in Pathogenicity of Three Different Forms of Rhizoctonia Inoculum and Assessment of Cultivar Resistance of Sugar Beet

Haque ME1 and Parvin MS2,3*

¹North Dakota State University, Fargo, USA

²Leibniz University Hannover, Germany

³Bangladesh Agricultural Research Institute, Joydebpur, Gazipur, Bangladesh

*Corresponding author: Most Shanaj Parvin, Leibniz University Hannover, Germany; Bangladesh Agricultural Research Institute, Joydebpur, Gazipur, Bangladesh

Received: July 02, 2021; Accepted: July 22, 2021; Published: July 29, 2021


Rhizoctonia solani causes pre-emergence and post-emergence damping-off, as well as crown and root rot of sugar beet (Beta vulgaris L.), which significantly affects the yield returns in the USA and Europe. The pathogen overwinters as sclerotia or melanized mycelium. Traditionally, the resistance of cultivars to R. solani is evaluated by scoring disease reactions at the crowns and roots of older seedlings, thus resistance is not evaluated during seed germination. Moreover, earlier studies evaluated cultivars resistance to R. solani using colonized whole barley or wheat grains which, unlike sclerotia, are artificial inocula of the pathogen that require time, space and technical know-how to produce. Moreover, colonized grains are prone to contamination with other pathogens, consumed by rodents/birds while applied in the field, and are often uneconomic. Considering those limitations, a study was undertaken (1) to develop in vitro methods to generate large-scale sclerotia, (2) to compare pathogenic potentials of sclerotia, mycelia, and colonized barley grains for optimization of dampingoff assays, and (3) to evaluate Rhizoctonia resistance of selected commercial cultivars during the seed germination phase. Comparing six different culture media, we found that R. solani had the highest radial growth (8.9 ± 0.04, cm³) at 8-days and the maximum number of sclerotia produced (203 ± 4.6) at 28-days in CV8 medium. We demonstrated significant differences in pathogenicity of the three different forms of R. solani inocula and susceptibility of cultivars to preand post-emergence damping-off. The highest pre-emergence damping-off and root rot were observed with sclerotia, and the highest post-emergence dampingoff was recorded with both sclerotial and colonized barley inocula. In addition, varietal differences in susceptibility to pre- and post-emergence damping-off were noted. The highest pre-emergence damping-off was recorded on cv Crystal 101RR and lowest in Maribo MA 504. The highest post-emergence damping-off was recorded on BTS 8500 and the lowest in Crystal 467. The maximum mean root rot was observed in BTS 8500, BTS 8606, and Crystal 101R. Our studies demonstrated that sclerotia serve as efficient natural inocula, reemphasized that host-pathogen interactions differ at the early vs. late stages of sugar beet growth, and highlighted the need to reevaluate commercial sugar beet cultivars for resistance at the seed germination stage.

Keywords: Sugar beet; Sclerotia; Mycelial plug; Colonized barley; Cultivars; Root rot


Sugar beet (Beta vulgaris L.) contributes approximately 20% of sugar worldwide [1] (International Sugar Organization, 2018). The rest is mainly derived from sugarcane. In the US, sugar beet contributes 55% of total sugar production [2]. From sowing to postharvesting, sugar beet is vulnerable to various biotic and abiotic stresses.

Many soil-borne pathogens, such as Rhizoctonia solani Kühn affect sugar beet stands and sugar yields. It is a genetically complex soil-borne fungus that causes pre-emergence and post-emergence damping-off, and also root and crown rot [3]. There are 13 anastomosis groups (AGs) of R. solani, which consist of AG 1 to AG 13; some AGs are host-specific while others have wide host ranges [4,5]. Sugar beet is prone to infection by the AG 2-2 strain [5]. The main AG subgroup that seriously impacts sugar beet yield is AG 2-2 IIIB [6,7]. However, the pathogenesis of this subgroup in sugar beet is not entirely well characterized. The primary inocula of the pathogen in nature are mycelia and sclerotia. This pathogen can survive in the soil for many years in the form of sclerotia, which is an undifferentiated aggregation of thick-walled melanized cells [8-10]. The sclerotia germinate under humid conditions and are often attracted by the root exudates of the germinated seedlings [11]. The host-pathogen interactions are generally initiated by mycelia that penetrate into the root cortex and cause infections to the tissue [12,13]. Rhizoctonia disease phenotyping in sugar beet fields often vary, primarily due to heterogeneous factors (biotic and abiotic), thus it is difficult to perform uniform disease trials [14]. To reduce variation in the experimental field, artificial inoculation using barley coated Rhizoctonia and greenhouse trials are usually performed as a complement to field trials. Preparing barley/wheat inocula requires time and space, technical know-how for large-scale production, as well as protection from other air-borne pathogens. Moreover, it can be difficult to evaluate resistance levels of sugar beet cultivars outdoors that provide a good correlation between artificial inoculum and disease severity. Since R. solani rarely produces any basidiospores, it is difficult to use the same amount and concentration of fungal biomass each time [15]. The utilization of sclerotia as inocula may circumvent those limitations. In an attempt to develop a uniform inoculation method and to better understand the R. solani-Beta vulgaris pathosystem, isolates of R. solani AG 2-2 IIIB were cultured in six different artificial media to study the morphology and development of sclerotia. Furthermore, infection severity was compared using three different forms of Rhizoctonia: sclerotia, mycelia, and colonized barley grains, in order to determine effective inoculation propagules and to evaluate the resistance response of the commercial cultivar in the greenhouse and in the field.

Materials and Methods

Fungal isolates of R. solani

Five R. solani isolates were collected from different diseased sugar beets from a field in Hickson, ND. Genomic DNAs (Norgen Biotek Corp, Fungi DNA Isolation Kit #26200) of the five isolates were used for Polymerase Chain Reaction (PCR) with the Internal Transcribed Spacer (ITS) [16]. Subsequently, PCR products were flushed by E.Z.N.A ®Cycle Pure Kit Omega Bio-tek, Norcross, GA) and four samples were sequenced by GenScript (Piscataway, NJ). The sequences were identical, and BLASTn analysis showed 100% sequence homology to R. solani AG 2-2 IIIB (Genbank accession: MN128569); thus, the five isolates were found to be clones of a single isolate. The isolate was maintained on CV8 and used for sub-culturing on six different media.

Culture media and development of sclerotia of R. solani

Microbial media play a significant role in the optimum mycelial growth and differentiation of different fungal species. The six different media: amended Clarified V8 (CV8), 50% Potato Dextrose Agar (PDA), 10% PDA, Methylene- Benomyl-Vancomycin (MBV), Cornmeal Agar (CMA) and Water Agar (WA), were prepared following the “Manual of Microbiological Culture Media”. The experimental design was a complete block design with four replications. Mycelial discs (3mm³) of R. solani, AG 2-2IIIB cut from the 7-day old mother colonies were transferred onto each of the six media in order to study the radial growth and development of sclerotia in vitro over time. Radial growth was measured using a digital caliper (Pittsburg 6” Composite DC, Item 93293) at four different time points: 2-day, 4-day, 6-day, and 8-day, respectively. Similarly, the number of sclerotia was counted at four-time points: 7-day, 14-day, 21-day, and 28-day, respectively. The experiment was repeated twice.

Categories of different sizes of sclerotia

Sclerotia were categorized into three different groups based on the size: large (≥4.00mm), medium (≥2.00mm), and small (≥0.5mm). To evaluate the size effect of sclerotia on causing disease, an experiment was conducted in the humidity chamber at 35°C and 85% relative humidity. Three different categories of sclerotia were used to inoculate Crystal 101RR with a complete block design of four treatments (including non-inoculated check) and four replications. The total number of observations was 16 (4-treatment × 4-replication). Ten seeds were sown in each treatment. The seedling stand count was recorded at two weeks post-inoculation.

In vitro inoculation on PDA using three different forms of R. solani inocula

To compare the efficacies of the three different inocula (i.e., sclerotia, mycelia, and colonized barley seeds), sugar beet seeds were co-cultured with respective inoculum in PDA with four replications. A non-inoculated check with four replications was used as a control. Sugar beet seeds were washed with 70% ethanol for 1 minute and rinsed twice with sterile water. Seeds were dried on sterile blotter paper under the laminar airflow cabinet. Three seeds (seed-to-seed distance 1cm) were placed with sterile forceps on each culture plate and a different one of the three inocula was placed close to each seed on the plate. Germination data were recorded from 2 days postinoculation (dpi) to 7 dpi. The experiment was repeated twice.

Greenhouse evaluation of cultivars’ susceptibility to Rhizoctonia inocula

Trials were conducted in a greenhouse at North Dakota State University, Fargo, North Dakota, USA. The experimental layout was a Complete Block Design (CRD) with seven sugar beet cultivars, four treatments, and four replications. The total number of observations was 112 with a split-plot arrangement. The seven commercial cultivars screened in this investigation had the 2018 Rhizoctonia root rot ratings in parenthesis, as follows: Crystal 101RR (4.50), Crystal 467RR (3.94), Hilleshog 4302RR (3.71), Maribo MA 504 (4.25), BTS 8606 (4.24), BTS8500 (4.36) and BTS80RR52 (3.96) (, Research Report 2018). Three different types of Rhizoctonia inocula: barley inoculum, sclerotia, and mycelia plug, as well as a non-inoculated check (autoclaved barley seed), were used for the inoculation of each cultivar. Plastic pots (27 x 13 x 13 cm3, T.O. Plastics, Inc.; Clearwater, MN, USA) were filled with vermiculite and perlite mixer (PRO-MIX FLX) amended with osmocote (15-9-12, 8˝ pot, 2.5 teaspoons per pot) fertilizer. Ten sugar beet seeds of each of the seven sugar beet cultivars were sowed into 2cm deep furrow and 1cm away from each seed in the center of each pot. Inoculation (one barley-inoculum/seed, one sclerotia/seed, one mycelial plug/seed, and mock inoculation) was done simultaneously with the sowed seed at the same depth and covered with mixer. The greenhouse temperature during the experiment was 25±2ºC during the day, with 80% relative humidity, and a 12-hour photoperiod. Plants were ensured water as needed to maintain adequate soil moisture conducive for plant growth and disease development.

Two weeks post-inoculation, the mean emergence and preemergence damping-off data were analyzed separately using splitplot R-package (Version 3.6.1, St. Louis, Missouri, USA). Percent post-emergence damping-off and stand counts data up to 42 dpi were also analyzed separately using split-plot R-package. At 56 dpi, plants were removed from pots, and roots were washed and rated for root rot disease severity using a modified 1-7 rating scale [17], where 1 = clean roots and no infection, 2 = <5% of root surface with black/brown symptoms, 3 = 5-25 % of root surface with black/brown symptoms; similarly, 4 = 26-50 % , 5 = 51-75 %, 6 = 75-100 % of root surface with black/brown symptoms, and 7 = dead plants (withered). The data were subjected to Analysis of Variance (ANOVA) and Fisher’s Protected Least Significant Difference was used to separate root rot severity means using the split-plot R-package software (3.6.1) (Table 7).

Statistical analyses

In both the in vitro and in vivo studies, the experiment was analyzed with the split-plot design using R-software (Version 3.6.1, St. Louis, Missouri, USA). Treatment means were distinguished by calculating Fisher’s Least significant difference at α = 0.05 confidence level.

Field evaluation of three different forms and rates of Rhizoctonia inocula

Field trials were conducted at Hickson, North Dakota, USA. The experimental layout was a Randomized Complete Block Design (RCBD) using a susceptible cultivar, Crystal 101 RR, with ten treatments (sclerotial number 1, 2, 3; mycelial disc number 1, 2, 3; colonized barley number 1, 2, 3; and non-inoculated check) and four replications (i.e., total number of observations was 40). The row length and width were 30 feet by 11 feet, respectively. Three different forms and rates of Rhizoctonia inocula were applied immediately after the seeds were sown.


Effect of different culture media and days on the radial expansion

Six different culture media were used to identify their effects on the radial growth of mycelia. There was a significant difference between the media, time points, interaction effects on the radial growth of mycelia, and the number of sclerotia. A mean comparison test was performed for radial growth and the number of sclerotia developed over different time points (Table 1 and Table 2).