Evaluation of mycoparasitic fungi as potential biological control agents of wattle rust (Uromycladium acaciae)

Abstract

Uromycladium acaciae (Cooke) P. Syd. & Syd., the causative agent of wattle rust, is an economically significant pathogen affecting black wattle (Acacia mearnsii De Wild) in the Republic of South Africa (RSA). Initial symptoms are brown powdery pustules on petioles, stems, and leaves which further develop into matting of leaves and pinnules, and rachis malformation. In severe cases, infection results in stunting of seedlings and mortality of young trees. Yield losses of up to 40% have been reported. The current control measures for wattle rust consist of azoxystrobin and triazole-based fungicides and the deployment of resistant or tolerant lines of A. mearnsii. However, due to undesirable environmental effects posed by fungicides, the substantial costs involved with both fungicide usage and the deployment of tolerant clones of A. mearnsii and increasing regulations around the use of fungicides in commercial forestry, there is a need for alternative control methods for wattle rust. The use of biological control presents a cost-effective and environmentally friendly alternative method for the management of wattle rust. Several mycoparasites of various rust diseases have been evaluated on different rust diseases as potential biological control agents. In this study, the mycoparasites Sphaerellopsis filum (Biv.- Bern. ex Fr.) Sutton [=Darluca filum (Biv.-Bern. ex Fr.) Berk.] and Akanthomyces uredinophilus Manfrino et al. were evaluated for biocontrol efficacy on wattle rust using an in vitro detached leaf assay. Leaves of black wattle infected with U. acaciae were collected from a local commercial plantation in Richmond, KwaZulu-Natal, South Africa. Rust-infected leaves were sprayed with conidial suspensions of 1 g.L-1 of S. filum (5.8x108 conidia.ml-1) and A. uredinophilus (1x106 conidia.ml-1) and incubated in plastic containers containing moist paper towels to create conditions of high humidity. Colonisation of rust pustules by A. uredinophilus and S. filum was assessed under a light microscope and a percentage colonisation for each treatment was estimated at four weeks post-inoculation. Colonised leaves were also viewed under an environmental scanning electron microscope (ESEM) to examine the interaction between the mycoparasitic fungi and U. acaciae. At four weeks post inoculation, A. uredinophilus and S. filum were able to colonise 41% and 44% of rust pustules, respectively. Under the ESEM, S. filum and A. uredinophilus demonstrated mycoparasitic activity on the teliospores of U. acaciae. Hyphae of S. filum and A. uredinophilus coiled around and punctured rust teliospores, causing them to rupture and deflate. Overall, conidia of both S. filum and A. uredinophilus were able to colonise and mycoparasitize U. acaciae teliospores. Optimal Integrated Pest Management (IPM) for disease suppression requires the seamless and coordinated integration of multiple compatible control strategies. Therefore, any biological agents that may be considered for use to suppress a disease should be compatible with the range of agrochemicals and adjuvants that are being used on the crop of interest, in order for the management strategy to be effective. To this end, the compatibility of two isolates of mycoparasitic fungi (Sphaerellopsis filum and Akanthomyces uredinophilus) and a commercial insect-pathogenic fungus (Beauveria bassiana, Eco-Bb®) with an insecticide (deltamethrin) and an adjuvant (Break-thru®) was evaluated. The experiment utilized an amended bioassay method with S. filum, A. uredinophilus, and B. bassiana being cultured on potato dextrose agar (PDA) amended with 250 μl.L-1, 125 μl.L-1 , and 500 μl.L-1 of Break-thru® , or amended with 1 ml.L-1, 0.5 ml.L-1 and 2 ml.L-1 of deltamethrin. Unamended media served as a negative control, whilst media amended with the commercial rust fungicide Amistar Top® served as a positive control. After three weeks, radial growth was measured from the centre of the fungal colony, and a percentage inhibition efficiency (IE) of Breakthru ®/ deltamethrin on the growth of the biological control agent was calculated. There was no significant inhibition in any of the biological agents by either Break-thru® or deltamethrin at all dosages tested. Therefore, S. filum, A. uredinophilus and B. bassiana are compatible with the insecticide (deltamethrin) and the adjuvant (Breakthru ®), and they could be combined in an integrated control program. Further evaluation of the two mycoparasitic fungi as potential biological control agents of wattle rust were undertaken in nursery pot trials. Pot Trial One investigated the biological control efficacy of mycoparasitic strains of S. filum and A. uredinophilus for control of wattle rust on three wattle varieties. Rust-infected cuttings of varieties PSO14, FW54 and SP644 were treated with S. filum and A. uredinophilus every two weeks for 12 weeks. They were also evaluated for rust levels every two weeks, as percentage leaf area infected. Experimental controls included a fungicide control (positive control) and untreated rust control (negative control). Bi-weekly applications of S. filum significantly reduced (p< 0.001) rust disease progression. Although applications with A. uredinophilus did reduce disease progression, it did not produce a significant reduction compared to the untreated rust control. After six and nine weeks of bi-weekly treatment applications, characteristic white mycelial growth of A. uredinophilus and S. filum, respectively, were observed on rust pustules. Characteristic spore morphology that resembled that of S. filum and A. uredinophilus was confirmed after re-isolation. There was no significant difference (p= 0.09605) between the wattle varieties in their response to the treatments. Overall, both S. filum and A. uredinophilus have the potential to control wattle rust. In Pot Trial Two, the optimal dosage and application frequency of S. filum and A. uredinophilus was evaluated on the FW54 (susceptible, frost tolerant) commercial wattle variety. Conidial suspensions of the mycoparasitic strains were prepared at dosages of 1 2 x, 1x and 2x at a quantity of 5x105 conidia.ml-1, 1x106 conidia.ml-1, and 2x106 conidia.ml-1 of A. uredinophilus, and 0.5 gram, 1 gram and 2 gram.L-1 of S. filum (5.8 x 108 conidia.ml-1). The conidial suspensions were sprayed at three frequencies which were once-off, bi-weekly (every two weeks), and monthly. Prior to every biweekly spray treatment, percentage rust severity (0 to 100%) was scored for all treatment frequencies on the fully expanded leaves directly below the new flush. The percentage rust severity was used to calculate the area under disease progress curve (AUDPC). For S. filum and A. uredinophilus, dosage and frequency of application had no significant interaction. All treatments reduced wattle rust relative to the untreated control treatment. Notably, the bi-weekly treatment with a 2x dosage of A. uredinophilus resulted in the least amount of rust disease, which was not significantly different to that of the fungicide treated control. In Pot Trial Three, the use of silicon fertilization in conjunction with A. uredinophilus (the best performing biocontrol treatment as identified in Pot Trial Two) for optimal control of wattle rust was evaluated. A spray application of 2x106 conidia.ml-1 of A. uredinophilus was combined with silicon fertilization at concentrations of 100, 200, and 400 mg.L-1 for evaluation of the control of wattle rust on the commercial wattle variety FW54. The A. uredinophilus spore suspension was sprayed bi-weekly (every two weeks), and treated seedlings were drenched every week with 10L of water containing silicon at 100, 200, and 400 mg L-1. Prior to every bi-weekly A. uredinophilus spray treatment, percentage rust severity (0 to 100%) was scored on the fully expanded leaves directly below the new flush. Experimental control treatments included a fungicide control (positive control, Amistar® Top, Syngenta) and an untreated rust control (negative control). At 16 weeks, leaf samples were taken from seedlings to evaluate the uptake and distribution of silicon using Field Emission Gun-Environmental Scanning Electron Microscopy (FEG-ESEM) and Energy Dispersive X-ray Spectroscopy (EDX). All treatments reduced wattle rust relative to the untreated control treatment. Treatment with A. uredinophilus + 400mg.L-1, silicon resulted in the least amount of rust disease. However, the A. uredinophilus + 0mg.L-1 silicon treatment also produced a significant reduction in rust. The EDX spectra and mapping showed that silicon was not taken up by treated plants. The combined application of A. uredinophilus and silicon fertilization did not result in the enhanced control of wattle rust because wattle appears not to take up silicon as a micronutrient. The overall results of this study suggest that both mycoparasites can be used as biological control agents for wattle rust. Bi-weekly applications of A. uredinophilus at 2x106 conidia.ml-1 was the best performing biocontrol treatment in these pot trials. However, it is unknown whether the nursery application of A. uredinophilus would yield sufficient fungal inoculum to control wattle rust under field conditions. Therefore, field trials should be implemented to establish the biocontrol efficacy of S. filum and A. uredinophilus under field conditions.