Evaluation of Cladosporium cladosporioides and Its Extracts for the Management of Pathogenic Bipolaris Species

Evaluation of in vitro interaction of Cladosporium isolates on B. oryzae, B. sorokiniana, and B. maydis.

A competitive bioassay was performed to evaluate the interaction of co-isolated Cladosporium and Bipolaris species on agar media plates to identify Cladosporium isolates with antagonistic effects on Bipolaris spp.  Twenty-one isolates of Cladosporium (Table 1) co-occurring with Bipolaris in the same lesion were co-plated against each three different Bipolaris spp. on an agar media. On the bottom Potato Dextrose Agar (PDA) media plates without added antibiotics, two intersecting straight lines were drawn resulting in four quadrants. Two plugs (2 cm diameter) of each Bipolaris and two plugs of each Cladosporium isolate were placed onto a PDA, in such a fashion that each species was placed onto opposite quadrants. PDA plates inoculated with only two plugs of Bipolaris were used as control plates. Inoculated media plates were incubated at 26°C in Percival Model I36VL incubator under 12-h light/dark condition for a week. Each combinations had three replications. After a week, the growth of Bipolaris colonies (colony diameter) was observed in each combination to determine any antagonistic interaction.

Cladosporium and Bipolaris interaction in PDA plates were mainly recorded using categorical scale inhibition zone used in Adhikari et al. 2022. Mainly two categories of interaction were observed: inhibition zone and colony contact. We defined inhibition zone as a distinct area between the two fungi where neither grows, resulting as Cladosporiumisolates restricting the colony growth of adjacent Bipolaris. These results were confirmed by comparing the colony size of Bipolaris-only plates. Inhibition zone were further categorized as strong or distinct zone (Usually >2 mm width) (IZ strong), and 2) weak or very thin zone (<2 mm width) (IZ weak). If a single inhibition zone was recorded if at least one interacting quadrant showed a strong or weak inhibition effect between two species. Similarly, colony contact was recorded when two species touched each other or grew in close proximity, basically with no inhibition zone. Within Cc, two different interactions were observed: growth of Cladosporium onto the Bipolaris colony (COG) and growth of Bipolaris onto the Cladosporium colony (BOC). Occasionally, some isolates of Cladosporium and Bipolaris colonies did not grow or expand compared to the control and other treatment combinations. In such case, it was difficult to confidently categorize the interaction, thus we recorded those interactions as not clear.

Table 1. List of Cladosporium isolates used against three different species of Bipolaris for competition assay.

Evaluation of the effect of Cladosporium isolates on disease severity of disease caused by B. sorokiniana and B. maydis.

Cladosporium isolates effective against Bipolaris in vitro were co-inoculated with each pathogenic Bipolaris spp. onto respective crop hosts rice, wheat and corn to evaluate the effectiveness of Cladosporium isolates in suppressing disease.

Wheat and corn seedlings were grown in the greenhouse at the University of Florida (UF), Gainesville Florida. Wheat cultivar “Jamestown” and corn line susceptible to B. maydis were used for the experiments. The seeds were grown in round plastic pots of 12.5 cm diameter containing potting mixture in the greenhouse at 20 to 25°C, 2-3 seeds per pot for wheat and 1 seed per pot for corn was used. Once the wheat seedlings emerged, they were thinned to one seedling per pot.  The wheat seedlings were grown up to 5 weeks and corn were grown for 4 weeks before they received the various co-inoculation treatments.

Five isolates of Cladosporium 1) Elymus seedling Clado (ElyS_C), 2) Citra _1 Clado (ElyC_C), 3) Guinea grass Clado (GG3_C), Cynodon Clado (Cyno_C) and 5) Rye Clado (Rye_C) were co-inoculated with Bipolaris sorokiniana isolate Citra 4 (BS) onto 5 weeks old wheat seedlings. From a week-old culture of BI and Cladosporium isolates, conidia were harvested using 0.1% Tween 20 (Sigma-Aldrich, St. Louis, MO, USA) sterile DI water. The conidia concentration was adjusted to 10⁵ conidia/ ml for BS and 3x10⁶ ml^-1 for all five isolates Cladosporium used in the experiment. There were 12 different inoculation treatments inoculated onto wheat: five Cladosporium isolates only treatments, one tween 20 water control, one BS only and five BS plus each Cladosporium isolate mixed 1:1 by volume. Each treatment had six replications and the experiment was repeated.  After inoculation, seedlings were bagged for 24 hours to maintain high relative humidity. One week post-inoculation, disease severity on each wheat seedling was recorded using the standard area diagram for spot blotch of wheat (Domiciano et al. 2014).

For co-inoculation of Cladosporium isolates and Bipolaris maydis (BM) on corn, all above mentioned five isolates of Cladosporium isolates were used against B. maydis isolates collected from an experimental corn field in Gainesville, Florida. The conidial suspension was the same concentration as mentioned earlier was prepared for the experiment. Similarly, there were 12 treatments inoculated onto four weeks old corn seedlings. Seedlings were bagged for 24 hours and after a week of inoculation, disease severity on corn seedling were recorded using the scale generated by Soliman et al. (2018). The experiment was conducted twice.

A combined analysis was conducted over the two experiments for each Bipolaris species. Data analysis was performed in JMP Pro version using a linear model. Fixed effects were experiments, inoculum treatments and their interaction. Disease severity data met the assumption of normality. Untransformed means and standard errors were presented in figures. Analysis of variance (ANOVA) was performed with P < 0.05 used for the significance test and Student’s HSD post hoc test at 95% confidence interval was used for pair-wise mean comparison among treatments.

Extraction and testing of chemical compounds with antifungal properties from Cladosporium isolate GG3_C against Bipolaris.

Five plugs each of 10 mm diameter GG3_C Cladosporium isolate from one-week old actively growing cultures on PDA plates were inoculated into 300 ml of potato liquid broth in volumetric flask. The liquid culture was allowed to grow for three weeks on a shaker at 100 rpm. Liquid extract was extracted using acetone and ethyl acetate filtration methods (Wang et al. 2013). The 300 mL of culture was centrifuged and the cell pellet was extracted with 10mL acetone. The acetone extract was mixed to the supernatant. The supernatant was extracted with twice volume of ethyl-acetate and concentrated under rotary evaporator. The concentrate was dissolved in 1 mL of methanol. The extracted samples were analyzed with analytical reverse phase HPLC (Agilent C18 column 250 x 4.6 mm, 0.5 mL/min; UV detection at 254) with a H₂O (0.1% FA)/ACN (0.1%FA). Further, LRMS analysis was used for optimizing the conditions for obtaining the specific mass fragments and data was recorded for validation of compounds. Low-resolution electrospray ionization mass spectrometry (LRESIMS) data were obtained on an Applied Biosystems MDS SCIEX 3200 Q TRAP LC/MS/MS system using the Turbo Spray ion source in positive ionization mode.

The remaining liquid extract was tested against pathogenic Bipolaris. Because the remaining liquid extract was not enough to test against all three Bipolaris, B. maydis was excluded as it has slower growth rate than B. oryzae and B.sorokiniana. The antagonistic assay was performed on a PDA agar media plate. A 2 mm diameter plug of Bipolaris was placed at the center of the plate and two holes (each 2mm diameter) were made on left and right side of centered Bipolaris plug in horizontal fashion. Each left and right holes were made at two different distances apart 0.5 cm and 1 cm apart from the center Bipolaris plug. Into each hole, 50 µL of liquid extract of GG3_C was transferred using pipette and the growth of pathogenic Bipolaris was recorded over 1, 3 5 and 11 days after inoculation of liquid extract. There were two replicates completed for each pathogenic Bipolaris species.