Termite–gut-associated actinomycetes as biological control agents against phytopathogen Pyrrhoderma noxium

Keywords: actinomycetes, antifungal agents, biological control, fermentation, microbial metabolism, Pyrrhoderma noxium, Streptomyces spp., termite guts.

Brown root-rot caused by phytopathogenic white-rot basidiomycete fungus, Pyyrhoderma noxium (P. noxium), formally known as Phellinus noxius¹ is a devastating disease prevalent in many tropical and subtropical countries.² It has caused much destruction throughout Southeast and East Asia, Oceania, the Pacific Islands, Africa, Central America, the Caribbean, and Australia.³ In fact, it has been one of the most damaging tree diseases in Taiwan,⁴ the Ryukyu Islands of Japan,⁵ Mariana Islands,⁶ Malaysia⁷ and Australia.⁸

This plant pathogen has a wide host range with over 260 susceptible tree species, represented by 59 families and can infect ornamental and agricultural crops as well as plantations and native forest trees.⁹ Within Australia, it occurs naturally throughout forests on the East Coast from Northern Queensland to Northern New South Wales where it has also caused issues for commercial plantation forests, fruit orchards and amenity plantings in urban areas.¹⁰ Identification of P. noxium causing death in avocado plantations was found in the Sunshine Coast hinterland, QLD in areas that were responsible for 50% of the total Australian avocado production.¹¹ Additionally, it has been reported impacting Hoop Pine plantations in QLD.¹² Currently, it is causing significant issues for the Brisbane area having killed many tree species throughout the greater Metropolitan area, including park and street trees located in Shorncliffe, Taringa, New Farm, Eagle Farm, West End, Hamilton, Indooroopilly, Brisbane River, and the City Centre. It has attacked many of the Brisbane trees such as figs, poinciana, jacarandas, Chinese elms, Moreton Bay eucalypts, kauris, and hoop pines and these include both public and privately owned trees.⁸

The infection cycle of this fungal disease begins in the roots where it commences colonisation and digestion of the tree’s wood. This degradation occurs through the production of digestive enzymes that can break down the vital components found in the cells of plants such as cellulose, hemicellulose and lignin.¹⁰ The decomposition of root-tissues leads to significantly reduced physical support and absorption of nutrients and water.² Symptoms include discolouration, bark exudations, defoliation, chlorosis, and dieback, however, these symptoms are usually observed in the later stage of infection when it is too late for intervention.² Transmission of this disease has two main modes: (1) through root-to-root contact with surrounding infected trees; and (2) infiltration and establishment of spores through entry points such as wounds on trees, exposed surfaces and on freshly cut tree stumps.¹⁰

Traditionally, chemical fungicides have been widely used for protection of plants against fungal pathogens;¹³ however, concerns surrounding the safety and environmental impacts of these chemicals has prompted investigations into alternative control methods.⁸ Recently, more emphasis has been placed on natural or biological approaches to controlling pests as it provides an environmentally friendly, safe, and inexpensive alternative for controlling plant pathogens. Utilisation of microorganisms and their secondary metabolites provides a promising alternative for disease prevention.¹⁴ Particularly, Streptomyces spp. are well known sources for secondary metabolism and their ability to produce various novel chemical structures with remarkable biological activities.¹⁵ Moreover, unexplored habitats and niches have attracted considerable attention in the discovery of specialised and active Streptomyces spp. such as microbiome symbionts.¹⁶

Specialised mutualistic relationships between microorganisms and hosts have seen the active culturing of beneficial microorganisms through co-evolution in exchange of bioactive small molecule.¹⁶ One such symbiosis exists between bacteria and the digestive tracts of insects, which contain communities of symbiotic and transient microorganisms.¹⁷ A relatively unexplored source is the guts of termites (Termitidae, Termitinae), which contain endosymbiotic protozoa and bacteria.¹⁸ Termites are insects belonging to the order Isoptera and predominantly feed on litter tissue and wood. It is difficult to digest lignocelluloses that are deficient in vitamins and other essential components. As a result, termites depend on beneficial symbiosis with a diverse flora of microorganisms within their hindguts for digestion and the acquisition of supplementary nutrition.¹⁹ Recent studies have revealed that actinomycetes which also cover the genus Streptomyces, were one of the dominant bacteria identified within the symbiotic lifestyle of termites.¹⁶ Actinomycetes aid termites, including nutrient recycling and exchange, as well as protecting the termites from invading pathogens.²⁰ It has been suggested that these unexplored insect-associated actinomycetes may house rare strains with the capacity to produce complex natural products with various biological activities.²¹ Although plant disease management by streptomycetes have been well documented,²² investigations into the use of termite–gut-associated ones against this pathogen are yet to be explored.

In the light of the above presented information, termite–gut-associated streptomycetes were investigated for their potential control of P. noxium. Two termite gut-associated streptomycetes (USC-595B, USC-596) were selected from the UniSC’s Microbial Library (Fig. 1).^23,24 First, a plate antagonism assay was performed, and all of the isolates were found to inhibit P. noxium growth (Fig. 2). Second, they were then fermented using solid-state fermentation on oatmeal agar and subsequent bioactive compound extraction was carried out as described by English et al. (2017).²⁵ Fermentation extracts obtained from the isolates were found to display antagonism towards the pathogen following the extract activity assays and scanning electron microscopical (SEM) observations (Figs 3, 4).

Fig. 1.  Phylogenetic tree of Streptomyces species based on 16S rRNA gene sequences in relation to known fungicide-producing Streptomyces.²⁵ The tree was constructed using the maximum likelihood method with sequences aligned by ClustalX2 on MEGA X. Kitasatospora setae (KF591080.1) was used as the outgroup.

Fig. 2.  Plate antagonism assay result (USC-595B).

Fig. 3.  Extract activity test (USC-595B).

Fig. 4.  SEM micrographs showing the growth of P. noxium in the presence of the fermentation extract of the Streptomyces sp. (USC-595B). (a) Untreated P. noxium showing healthy hyphae. (b–d) Collapsed hyphae after exposure to the extract. Bars indicate: (a) 100 µm; (b) 100 µm; (c) 50 µm; and (d) 20 µm.

Structure elucidation of the bioactive compounds are underway at the UniSC. However, preliminary findings indicate that insect microbiome-symbionts might offer a feasible source for biological control of Pyrrhoderma noxium. Field trials are also underway to determine the effectivity of the isolates and their compounds under environmental conditions to complement already used Trichoderma biological control applications by the Brisbane City Council.²⁶

Fig. 1 contains accession numbers for the molecular data, and data is presented in the images (Figs 2–4).

The authors declare no conflicts of interest.

The substudy presented here is part of an ongoing P. noxium management research using biological control agents jointly conducted by the UniSC and Brisbane City Council (BCC). Both parties provide cash and in-kind contributions.