Inoculation and Screening Methods for Major Sorghum Diseases Caused by Fungal Pathogens: Claviceps africana, Colletotrichum sublineola, Sporisorium reilianum, Peronosclerospora sorghi and Macrophomina phaseolina

Abstract

Sorghum is the fifth most important crop globally. Researching interactions between sorghum and fungal pathogens is essential to further elucidate plant defense mechanisms to biotic stress, which allows breeders to employ genetic resistance to disease. A variety of creative and useful inoculation and screening methods have been developed by sorghum pathologists to study major fungal diseases. As inoculation and screening methods can be keys for successfully conducting experiments, it is necessary to summarize the techniques developed by this research community. Among many fungal pathogens of sorghum, here we summarize inoculation and screening methods for five important fungal pathogens of sorghum: Claviceps africana, Colletotrichum sublineola, Sporisorium reilianum, Peronosclerospora sorghi and Macrophomina phaseolina. The methods described within will be useful for researchers who are interested in exploring sorghum-fungal pathogen interactions. Finally, we discuss the latest biotechnologies and methods for studying plant-fungal pathogen interactions and their applicability to sorghum pathology.

Keywords: Claviceps africana; Colletotrichum sublineola; Macrophomina phaseolina; Peronosclerospora sorghi; Sporisorium reilianum; disease evaluation; disease screening; fungal pathogens; pathogen inoculation; sorghum.

Figure 1

Illustrations of the three secondary conidia inoculation techniques described by Tonapi et al. [9]. (a) Spray inoculation of secondary conidia (concentration: 1 × 10⁴ conidia/mL). (b) Stigmas (wet or dry) were brushed with secondary conidia collected from the lids of inverted agar plates. (c) Secondary conidia were applied using compressed air.

Figure 2

Illustrations of the dip inoculation and sponge inoculation methods. (a) Panicles were dipped into inoculum with approximately 1 × 10⁶ conidia/mL [11]. (b) A sponge soaked with C. africana spore suspension is physically in contact with panicles [12].

Figure 3

Illustrations of the spray inoculation and excised-leaf inoculation methods (a) 1 × 10⁶ conidia/mL of inoculum was sprayed and colonized sorghum seeds were dropped into the leaf whorl at 8 leaf stage sorghum plants [14]. (b) Excised leaves on ½ PDA plates were droplet inoculated with 1 × 10⁶ conidia/mL of inoculum [19]. Compared to the typical spray inoculation method, the excised leaf assay shortens the waiting time for screening as the leaves can be scored at 4 dpi. Created with BioRender.com (accessed on 11 January 2023).

Figure 4

Screening methods for an excised-leaf assay. (a) Typical screening method based on the presence or absence of acervuli formation at 4 dpi [19]. This binary system is useful for quick screening of host resistance, where plants are considered susceptible if acervuli are present and resistant if acervuli are absent. (b) A modified screening method using a 1–5 scale [21]. This system provides more resolution for scoring the severity of infection. The scale ranges from 1 (no infection) to 5 (>5 acervuli perfectly formed), with intermediate scores for different levels of infection.

Figure 5

Three different inoculation methods of S. reilianum to sorghum. (a) Teliospores mixed with soil followed by planting seeds, (b) Hypodermic injection of sporidial suspension to sorghum seedlings and (c) Seedling inoculation with spore suspension in agar.

Figure 6

Illustrations show screening methods for S. reilianum in greenhouse & field and excised-leaf assays. (a) Screening is based on the presence or absence of infected heads in plants with fully developed grains and no sori. (b) Screening for the seedling agar inoculation method is based on the presence or absence of dark or brown spots on the first leaf.

Figure 7

Six P. sorghi inoculation methods for sorghum described by Narayana et al. [34]. (a) Sandwich inoculation of sprouted seeds placed between two infected leaf pieces on a wet Whatman filter paper in a Petri plate. (b) Spray inoculation of sprouted seeds with a conidial suspension. (c) Dip inoculation of sprouted seeds immersed in a conidial suspension. (d) Drop-inoculated seedlings at the one-leaf stage using a micro-syringe to place a droplet of inoculum in the whorl of the first leaf. (e) Spray inoculation of seedlings at the one-leaf stage with a conidial suspension. (f) Conidial showering of seedlings by covering the outer rim of each pot with moist muslin cloth and a layer of detached downy mildew-infected leaves, allowing the conidia to drop onto the emerging seedlings.

Figure 8

Overview of the sick-plot method. Black masses in soil represent the microsclerotia of M. phaseolina. (a) tested genotypes in green, light green corresponds to susceptible checks. (b) Moisture stress induction. (c–f) Data recording for screening purposes. Created with BioRender.com (accessed on 11 January 2023).

Figure 9

Illustrations for the toothpick method. (a) Wooden toothpicks are submerged in PDB. (b) Wooden toothpicks in PDB are sterilized. (c) Toothpicks are inoculated with M. phaseolina and incubated for ten days at 25 ± 1 °C. (d) Infested toothpicks are placed into the second internode. (e,f) Sampling and evaluating inoculated plants. 4 Orange crosses indicate inoculated plants. Created with BioRender.com (accessed on 11 January 2023).

Figure 10

The Phaseolinone sensitivity screening procedure. (a) The procedure starts with the inoculation of sterilized PDB with M. phaseolina in a capped Erlenmeyer flask (b) The culture is then shaken for 8–9 days at 150 rpm and 25–30 °C. (c) Afterward, the culture is filtered through a Whatman filter paper No.1 to harvest the filtrate. (d) The harvested filtrate is directly used to test the genotypes or serve as a substrate to purify the phaseolinone. (e) A serial dilution of the culture is conducted to prepare known concentrations of the raw CFCF or purified phaseolinone. (f) The seedlings of the tested genotypes are then inoculated with the prepared dilutions of M. phaseolina. (g) Evaluations for diseases are recorded at 10 dpi. Created with BioRender.com (accessed on 11 January 2023).

Figure 11

Inoculum preparation for charcoal rot greenhouse evaluation. (a) M. phaseolina is grown on PDA and then blended at high speed for 10 min using a Waring blender. (b) The suspension is filtered through four layers of sterile cheesecloth to obtain small fragments. (c) The resulting suspension is centrifuged, and the pellet is resuspended in 50 mL of PBS. (d) The concentration is adjusted to 5 × 10⁴ hyphal fragments per milliliter by diluting the suspension using PBS. (e) The suspension goes through multiple steps of dilution and is spread onto a PDA plate. (f) The PDA plates are incubated at 30 °C for three days, and (g) the colony-forming units are counted for ten replicate plates. (h) Inoculation is performed on sorghum plants 14 days after flowering by injection. Created with BioRender.com (accessed on 11 January 2023).