Native and Non-Native Soil and Endophytic Trichoderma spp. from Semi-Arid Sisal Fields of Brazil Are Potential Biocontrol Agents for Sisal Bole Rot Disease

Abstract

Sisal (Agave sisalana) bole rot caused by Aspergillus welwitschiae is the main phytosanitary problem affecting sisal in the Brazilian semi-arid region. The aim of this study was to evaluate Trichoderma spp. as biocontrol agents for sisal bole rot. Native and non-native species, both soil inhabitants and endophytes, and isolated from different plant hosts were tested. Anatomical studies of the interaction among A. sisalana, Trichoderma spp., and A. welwitschiae were performed. T. cf. asperellum (isolate F12), an endophyte of sisal leaves; T. cf. asperellum (TCS83) from banana plant soil; T. lentiforme (TCS15) and T. harzianum (species complex) (TCS35 and TCS76) from sisal root soil; T. spirale (R62) and T. saturnisporum (R75), endophytes of sisal roots, were the most efficient isolates, with inhibition of A. welwitschiae mycelial growth by up to 70%, and inhibition of sporulation and spore germination by 99%. A reduction in disease incidence of 70 to 93% and in disease severity of 97% was achieved. T. lentiforme (TCS1), T. harzianum (species complex) (TCS35 and R72), and T. koningiopsis (R78) showed mycoparasitism. An increase in cell wall thickness of bole tissue colonized by these Trichoderma species indicated that induced plant defense responses occurred, preventing pathogen colonization, which should be further investigated. Native and non-native Trichoderma species can control sisal bole rot disease.

Keywords: Agave sisalana; Aspergillus welwitschiae; Trichoderma cf. asperellum; Trichoderma harzianum (species complex).

Figure 1

Colony diameter of Aspergillus welwitschiae paired with Trichoderma spp. in PDA medium. Bars with same letter do not differ statistically by the test of Scott–Knott (p ≤ 0.05).

Figure 2

Interaction between isolates of Trichoderma and A. welwitschiae in paired cultures. Except for the control with A. welwitschiae only, all paired cultures have Trichoderma on the left side and A. welwitschiae on the right side.

Figure 3

Interaction between isolates of Trichoderma and A. welwitschiae, observed under a microscope with 1000×. (A,B) Unstained slides showing Trichoderma growth and coiling around the hyphae of A. welwitschiae, with hyphae strangulation. (C,D) Stained slides showing Trichoderma on A. welwitschiae’s hyphae, with the formation of appressorium-like structures and penetration into A. welwitschiae’s hyphae. Scale: 50 µm (A,B); 20 µm (C,D).

Figure 4

Colony diameter (cm) and sporulation (number of spores per cm² of colony) of A. welwitschiae affected by volatile compounds produced by Trichoderma spp. Bars with the same letters do not differ statistically by the test of Scott–Knott (p ≤ 0.05).

Figure 5

A. welwitschiae exposed to volatile compounds produced by Trichoderma spp. The culture with A. welwitchiae without Trichoderma spp. refers to the control treatment.

Figure 6

Inhibitory effect of non-volatile compounds produced by Trichoderma spp. on colony diameter (cm), sporulation (number of spores per cm² of colony), and spore germination (%) of A. welwitschiae. Bars with the same letters do not differ statistically by the test of Scott–Knott (p ≤ 0.05).

Figure 7

Disease index of sisal bole rot in plants treated with T. harzianum (species complex) (TCS35) and T. lentiforme (TCS15) and inoculated with A. welwitschiae (AW). These were compared to plants without Trichoderma but inoculated with A. welwitschiae (AW), plants with wounded bole tissue but treated only with Trichoderma, and those that received solely distilled water (DW).

Figure 8

Sisal plants at 30 days after inoculation with A. welwitschiae. (A) Plants inoculated only with A. welwitschiae (control treatment) showing complete bole and leaf base rot, with plant death. (B) Plants treated with T. harzianum (species complex) (TCS35) and inoculated with A. welwitschiae.

Figure 9

Sisal bole rot incidence (%) and disease index (%) in sisal plants treated with Trichoderma spp. and inoculated with the pathogen A. welwitschiae. Means followed by the same letter in the same column are not statistically different, by Scott–Knott (p ≤ 0.05).

Figure 10

Heat map with a dendrogram of in vitro tests for control of sisal bole rot with Trichoderma spp., as well as disease incidence and severity index of sisal plants treated with Trichoderma spp. and Aspergillus welwitschiae. The dendrogram on the left side depicts relationships between disease incidence and severity index, whereas the dendrogram on top depicts the relationships between Trichoderma spp. and A. welwitschiae. Dark red indicates higher values while dark blue indicates lower values. NVS—non-volatile compounds and sporulation. VS—volatile compounds and sporulation; PCD—paired colony diameter; VCD—volatile compounds and colony diameter; DIncid—disease incidence; DIndex—disease index; NVCD—non-volatile compounds and colony diameter; NVSG—non-volatile compounds and spore germination.

Figure 11

Scores plot and two component analysis of the in vitro tests for biocontrol of sisal bole rot with Trichoderma, as well as disease incidence and severity index of sisal plants treated with different isolates of Trichoderma and with Aspergillus wewitschiae.

Figure 12

Transversal sections of sisal bole inoculated with Trichoderma spp. and A. welwitschiae. (A) Healthy sisal bole tissue (noinoculation). (B) Sisalbole tissue inoculated with Trichoderma and A. welwitschiae. (C,D) Note the presence of massive A. welwitschiae hyphae (arrow) in infected and rot bole tissue. (E) Parenchyma cells with increased thickness of cell walls (arrow). Tissue double-stained with toluidine blue and basic fuchsin; the blue to green color indicates an affinity for acid structures such as lignin deposited in the cell walls. (F,G) Lignification of parenchyma cellsaround damaged bole tissue folowing infection by A. welwitschiae. Samples stained with toluidine blue; cell walls rich in pectin exhibit purple color, cellulosic cell walls stain blue, and lignified cell walls can exhibit blue to green colors. Abbreviations: Aw—Aspergillus welwitschiae; LC—Lignified Cells; P—Parenchyma; DT—Dead Tissue. Disease Scale: 150 µm (F); 60 µm (A,B); 1 µm (C–E,G).