agronomy Case ReportReport Biocontrol of Crown Gall byby RhizobiumRhizobium rhizogenesrhizogenes:: Challenges in Biopesticide Commercialisation 1 2, Allen Kerr 1 and Gary Bullard 2,** 1 Department of Plant Pathology, University of Adelaide, Adelaide, SA 5064, Australia; [email protected] 1 Department of Plant Pathology, University of Adelaide, Adelaide SA 5064, Australia; [email protected] 2 Bio-Care Technology Pty Ltd., Myocum, NSW 2481, Australia 2 Bio-Care Technology Pty Ltd., Myocum NSW 2481, Australia * Correspondence: [email protected] * Correspondence: [email protected] Received: 18 June 2020 2020;; Accepted: 30 July 2020 2020;; Published: 3 3 August August 2020 2020 Abstract: The biocontrolbiocontrol of crown gall has been practisedpractised in AustraliaAustralia for 4848 years.years. Control is so eefficientfficient thatthat itit is is di difficultfficult to to find find a galleda galled stone stone fruit fruit tree, tree, when when previously, previously, crown crown gall had gall been had abeen major a problem.major problem. This paper This explainspaper explains how it works how andit works why onlyand pathogenswhy only arepathog inhibited.ens are A inhibited. commercial A biopesticidecommercial biopesticide is available inis Australia,available in Canada, Australia, Chile, Canada, New Zealand,Chile, New Turkey, Zealand, the USA, Turkey, South the Africa USA, andSouth Japan. Africa The and challenges Japan. The of commercialisingchallenges of commercialising a biopesticide area biopesticide outlined. Rigid are outlined. regulations Rigid are preventingregulations theare widerpreventing use of the biocontrol wider use organisms. of biocontrol organisms. Keywords: crown gall; Agrobacterium; Rhizobium; agrocin 84; TM84; agrocinopine; biopesticidebiopesticide 1. Introduction 1. Introduction Crown gall is caused by soil-inhabiting bacteria that transfer some of their DNA to plant cells [1], Crown gall is caused by soil-inhabiting bacteria that transfer some of their DNA to plant cells which, as a result, grow and divide out of control to form galls or tumours. The tumour-inducing [1], which, as a result, grow and divide out of control to form galls or tumours. The tumour-inducing genes are located on a plasmid, pTi [1]. A recent review describes, in detail, the infection process [2]. genes are located on a plasmid, pTi [1]. A recent review describes, in detail, the infection process [2]. Infection may occur through wounds or lenticels [3]. An efficient biocontrol agent should be able to Infection may occur through wounds or lenticels [3]. An efficient biocontrol agent should be able to colonise both sites. colonise both sites. 2. Biocontrol 2. Biocontrol In Australia, crown gall (Figure1) of several plant species has been controlled biologically since In Australia, crown gall (Figure 1) of several plant species has been controlled biologically since the 1970s [4]. The control organism is strain K84, a biovar 2 strain of Agrobacterium radiobacter (now the 1970s [4]. The control organism is strain K84, a biovar 2 strain of Agrobacterium radiobacter (now Rhizobium rhizogenes) and an efficient root coloniser [5]. Rhizobium rhizogenes) and an efficient root coloniser [5]. Figure 1. Crown g gallall on rose (provided by Melodie Putnam). Agronomy 2020, 10, x; doi: FOR PEER REVIEW www.mdpi.com/journal/agronomy Agronomy 2020, 10, 1126; doi:10.3390/agronomy10081126 www.mdpi.com/journal/agronomy Agronomy 2020, 10, 1126 2 of 11 Agronomy 2020, 10, x FOR PEER REVIEW 2 of 10 Initially,Initially, the inoculant the inoculant was wassupplied supplied by the by scientific the scientific laboratory laboratory that isolated that isolated K84, but K84, later, but a later, commerciala commercial product, product, containing containing strain strainK1026, K1026, a modified a modified version version of strain of strain K84, K84,was wasproduced produced [6] [6] and andregistered registered as a asbiopestici a biopesticidede in 1988 in 1988 (Australia), (Australia), 2000 2000 (USA) (USA) and and2005 2005 (Turkey). (Turkey). Control Control occurs occurs throughthrough the theproduction production of an of anantibiotic, antibiotic, agrocin agrocin 84 84[7] [7 (Figure] (Figure 2).2). It It is is an an adenine nucleotidenucleotide withwith two two substituents,substituents, oneone ofof which which controls controls uptake uptake and and the the other, other toxicity, toxicity [8]. [8]. Agrocin Agroci 84n prevents84 prevents protein proteinsynthesis synthesis by inhibitingby inhibiting the the essential essential enzyme enzyme leucyl leucyl tRNA tRNA synthetase synthetase [9,10 [9,10]. ]. Figure 2. N 6 d Figure 2. AgrocinAgrocin 84 in 84N- in6β-D-Gβlucopyranosyl- -Glucopyranosyl-form-form (provided (provided by the by late the Max late MaxTate). Tate). Genes for the synthesis of agrocin 84 are located on a plasmid, pAgK84 [11], a conjugative plasmid Genes for the synthesis of agrocin 84 are located on a plasmid, pAgK84 [11], a conjugative that can be transferred to other strains, including pathogens [12]; it also carries immunity to agrocin plasmid that can be transferred to other strains, including pathogens [12]; it also carries immunity to 84 [13,14]. To prevent transfer, the transfer genes were located [13] and a transfer-minus deletion agrocin 84 [13,14]. To prevent transfer, the transfer genes were located [13] and a transfer-minus mutant was constructed [6] to produce strain K1026. As restriction enzymes and a ligase were used deletion mutant was constructed [6] to produce strain K1026. As restriction enzymes and a ligase to construct K1026, it was originally designated a GMO, a genetically modified organism. However, were used to construct K1026, it was originally designated a GMO, a genetically modified organism. as it is a deletion mutant and contains no foreign DNA, it is no longer considered as such in most However, as it is a deletion mutant and contains no foreign DNA, it is no longer considered as such countries. The system has at least two very interesting features: only pathogens are inhibited by the in most countries. The system has at least two very interesting features: only pathogens are inhibited antibiotic so there is no damage to the environment, and the system has been used commercially for by the antibiotic so there is no damage to the environment, and the system has been used about 48 years but there is no evidence of resistance to agrocin 84 developing. Control is so effective commercially for about 48 years but there is no evidence of resistance to agrocin 84 developing. that in Australia, it is difficult to find crown gall on roses and stone fruit trees when once, it was a Control is so effective that in Australia, it is difficult to find crown gall on roses and stone fruit trees major problem. The biocontrol of crown gall probably contributes about 100 million Australian dollars when once, it was a major problem. The biocontrol of crown gall probably contributes about 100 (AUD) annually to the Australian economy. million Australian dollars (AUD) annually to the Australian economy. Crown galls contain unusual compounds, now called opines, found nowhere else in the plant Crown galls contain unusual compounds, now called opines, found nowhere else in the plant kingdom [15]. They are used as nutrients by the inciting bacteria, and they alone can utilise them. kingdom [15]. They are used as nutrients by the inciting bacteria, and they alone can utilise them. Agrocin 84 is a Trojan Horse antibiotic because it uses a bacterial uptake system designed for the uptake Agrocin 84 is a Trojan Horse antibiotic because it uses a bacterial uptake system designed for the of an opine, agrocinopine A [16,17] (Figure3). uptake of an opine, agrocinopine A [16,17] (Figure 3). Agronomy 2020, 10, 1126 3 of 11 Agronomy 2020, 10, x FOR PEER REVIEW 3 of 10 FigureFigure 3. 3.AgrocinopineAgrocinopine A A(provided (provided by by Maarten Maarten Ryder). Ryder). TheThe genetics genetics of of the the system havehave been been established established by by Stephen Stephen Farrand’s Farrand’s group group [18], [18] and, theand uptake the uptakemechanism, mechanism by the, by Morera the Morera and Reader and Reader labs [19 ],labs where [19], a where pyranase-2-phosphate a pyranase-2-phosphate motif, present motif, in presentboth agrocin in both 84 agrocin and agrocinopine 84 and agrocinopine A, is all important.A, is all important. As opines As can opines only becan utilised only be by utili crownsed by gall crownpathogens, gall pathogens only pathogens, only pathogens are inhibited. are inhibited. Agrocin 84Agrocin is a pro-drug 84 is a pro because-drug afterbecause entering after entering a cell, it is a processedcell, it is processed to remove to the remove sugar [the9,10 sugar] and form[9,10] a and toxic form moiety, a toxic TM84. moiety, Why TM84. resistance Why has resistance not developed has notis developed unknown, is although unknown, the although fact that strainsthe fact K84that and strains K1026 K84 also and produce K1026 also agrocin produce 434 [agrocin20] might 434 be [20]significant. might be Thesignificant. commercial The biopesticidecommercial controlsbiopesticide crown controls gall on crown stone fruits, gall on nut stone trees, fruits, roses nut and trees, several rosesother and crops several but not other all, notablycrops but on grapevinesnot all, notably where on crown grapevines gall is caused where by crown strains gall resistant is caused to agrocin by strains84. Attempts resistant to to develop agrocin a84 commercial. Attempts to biocontrol develop a for commercial grapevine biocontrol crown gall for continue grapevine [21,22 crown]. gall continueIt should[21,22]. be noted that in crown gall, the control organism is closely related to the pathogen. PerhapsIt should more be attentionnoted that should in crown be paidgall, the to closecontrol relatives organism when is closely seeking related new to biopesticides the pathogen. [23 ]. PerhapsImportantly, more theattention identification should of be agrocin paid 84to asclose a potent relatives leucyl when tRNA seeking synthetase new inhibitor biopesticides has heralded [23].