Early Physiological Plant Response and Systemic Effects of Huanglongbing Infection in Split-Root Plants.

Huanglongbing (HLB), caused by 'Candidatus Liberibacter asiaticus' (CLas), is a devastating disease of citrus. After initial infection, CLas quickly colonizes the root system before canopy symptoms develop. There is limited understanding of CLas movement from roots to canopy and local and systemic effects on root dynamics. Using split-root rhizoboxes and late summer below-the-split bud inoculation, effects of local infection on systemic disease development were studied. Upward bacterial movement from roots is linked to seasonal flushes and CLas population in roots. CLas stayed isolated to one side of the roots for at least 8 months, until the spring flush. HLB caused differential root responses depending on tree age at infection. Systemic effects, independent of CLas movement, occur very early after infection. Stimulation of root growth occurred on noninfected roots prior to CLas detection in 1.5-year-old trees but decreased in 2.5-year-old trees. Independent of tree age, root growth was stimulated during spring root flushes after CLas population stabilized. Root dieback began simultaneously with detection of CLas in roots (6 weeks postinoculation). Infection and tree age altered root lifespan. In total, 1.5-year-old CLas-infected roots from summer and fall flushes had 3 and 6 weeks reduced lifespan. In contrast, 2.5-year-old CLas-infected plants lifespan was unaffected. Season affected root lifespan with late summer root flush lifespan was three times shorter than fall or spring root flushes. Split-root inoculation allowed study of local and systemic effects of CLas infection in roots, information crucial to prolonging the productivity of HLB-affected trees.

Assessment of the Effect of Thermotherapy on 'Candidatus Liberibacter asiaticus' Viability in Woody Tissue of Citrus via Graft-Based Assays and RNA Assays.

In 2019, citrus production in Florida declined by more than 70%, mostly because of Huanglongbing (HLB), which is caused by the bacterium 'Candidatus Liberibacter asiaticus' (CLas). Thermotherapy for HLB-affected trees was proposed as a short-term management solution to maintain field productivity. It was hypothesized that thermotherapy could eliminate HLB from affected branches; therefore, the study objectives were to show which time-temperature combinations eliminated CLas from woody tissues. Hardening, rounded Valencia twigs collected from HLB-affected field trees were treated in a steam chamber at different time-temperature combinations (50°C for 60 s; 55°C for 0, 30, 60, 90, and 120 s; 60°C for 30 s; and an untreated control). Three independent repetitions of 13 branches per treatment were grafted onto healthy rootstocks and tested to detect CLas after 6, 9, and 12 months. For the RNA-based CLas viability assay, three branches per treatment were treated and bark samples were peeled for RNA extraction and subsequent gene expression analyses. During the grafting study, at 12 months after grafting, a very low frequency of trees grafted with twigs treated at 55°C for 90 s and 55°C for 120 s had detectable CLas DNA. In the few individuals with CLas, titers were significantly lower (P ≤ 0.0001) and could have been remnants of degrading DNA. Additionally, there was a significant decrease (P ≤ 0.0001) in CLas 16S rRNA expression at 55°C for 90 s, 55°C for 120 s, and 60°C for 30 s (3.4-fold change, 3.4-fold change, and 2.3-fold change, respectively) in samples 5 days after treatment. Heat injury, not total CLas kill, could explain the limited changes in transcriptional activity; however, failed recovery and eventual death of CLas resulted in no CLas detection in most of the grafted trees treated with the highest temperatures or longest durations.

Responses of Anastrepha suspensa, Diachasmimorpha longicaudata, and Sensitivity of Guava Production to Heterorhabditis bacteriophora in Fruit Fly Integrated Pest Management.

Caribbean fruit fly, also known as Caribfly or Anastrepha suspensa , is a major tephritid pest of guavas. A virulent entomopathogenic nematode (EPN) species was investigated to suppress the fruit-to-soil stages of Caribflies, which are also attacked by the koinobiont parasitoid Diachasmimorpha longicaudata in south Florida. The main objective was to develop a feasible and cost-effective EPN-application method for integrated pest management (IPM) of Caribfly to improve guava production. Naturally infested guavas were treated with increasing Heterorhabditis bacteriophora infective juvenile (IJ) concentration or rate (0, 25, 50, …, 1,600 IJs cm -2 ) in field trials to measure the optimum IJ rate and then examine sensitivity of producing guavas to inclusion of Heterorhabditis bacteriophora in Caribfly IPM plans. Relative survival of Caribfly in treatments significantly decreased with increasing IJ rate from 0 to 100 IJs cm -2 . Similarly, probability of observing large numbers of parasitoid wasps ( Diachasmimorpha longicaudata ) in EPN treatments significantly declined with increasing IJ rate (0-100 IJs cm -2 ), even though the non-target effects of Heterorhabditis bacteriophora on relative survival of Diachasmimorpha longicaudata could not be determined because of few emerging parasitoid wasps. Optimum suppression (⩾ 60%) of Caribfly was consistently achieved at 100 IJs cm -2 or 17,500 IJs fruit -1 . Profitability analysis showed that Heterorhabditis bacteriophora can be included in Caribfly IPM tactics to produce guavas. Costs of EPNs in Caribfly IPM are minimized if Heterorhabditis bacteriophora is strategically applied by spot treatment of fruit. Repayment of costs of EPN-augmentation by spot treatments appears achievable by recovering 5.71% of the annual yield losses (⩾1,963 kg ha -1 ≈ US$ 8,650 ha -1 ), which are largely due to Caribfly infestation. Hectare-wide EPN-augmentation (or broadcasting) method requires more fruit recovery than the total annual yield losses to repay its high costs. Profitability of guava production in south Florida will not be very sensitive to marginal costs of the spot treatment method, when compared to the field-wide broadcasting of Heterorhabditis bacteriophora .

Cytochrome P450­catalyzed L-tryptophan nitration in thaxtomin phytotoxin biosynthesis.

Thaxtomin phytotoxins produced by plant-pathogenic Streptomyces species contain a nitro group that is essential for phytotoxicity. The N,N'-dimethyldiketopiperazine core of thaxtomins is assembled from L-phenylalanine and L-4-nitrotryptophan by a nonribosomal peptide synthetase, and nitric oxide synthase-generated NO is incorporated into the nitro group, but the biosynthesis of the nonproteinogenic amino acid L-4-nitrotryptophan is unclear. Here we report that TxtE, a unique cytochrome P450, catalyzes L-tryptophan nitration using NO and O(2).

Viability of 'Candidatus Liberibacter asiaticus' prolonged by addition of citrus juice to culture medium.

Huanglongbing, or citrus greening disease, is associated with infection by the phloem-limited bacterium 'Candidatus Liberibacter asiaticus'. Infection with 'Ca. L. asiaticus' is incurable; therefore, knowledge regarding 'Ca. L. asiaticus' biology and pathogenesis is essential to develop a treatment. However, 'Ca. L. asiaticus' cannot currently be successfully cultured, limiting its study. To gain insight into the conditions conducive for growth of 'Ca. L. asiaticus' in vitro, 'Ca. L. asiaticus' inoculum obtained from seed of fruit from infected pomelo trees (Citrus maxima 'Mato Buntan') was added to different media, and cell viability was monitored for up to 2 months using quantitative polymerase chain reaction in conjunction with ethidium monoazide. Media tested included one-third King's B (K), K with 50% juice from the infected fruit, K with 50% commercially available grapefruit juice, and 100% commercially available grapefruit juice. Results show that juice-containing media dramatically prolong viability compared with K in experiments reproduced during 2 years using different juice sources. Furthermore, biofilm formed at the air-liquid interface of juice cultures contained 'Ca. L. asiaticus' cells, though next-generation sequencing indicated that other bacterial genera were predominant. Chemical characterization of the media was conducted to discuss possible factors sustaining 'Ca. L. asiaticus' viability in vitro, which will contribute to future development of a culture medium for 'Ca. L. asiaticus'.

qPCR Quantification of Pathogenic Guignardia citricarpa and Nonpathogenic G. mangiferae in Citrus.

Citrus black spot, a major citrus disease caused by Guignardia citricarpa, was recently introduced in Florida. The nonpathogenic fungal endophyte G. mangiferae is commonly found in the same citrus tissues as G. citricarpa. Quantitative polymerase chain reaction (qPCR) assays based on internal transcribed spacer (ITS)-1 genes were developed to detect, quantify, and distinguish between these morphologically similar organisms in environmental samples. The primer/probe sets GCITS and GMITS were more than 95% efficient in single-set reactions in complex environmental DNA samples. Detection of 10 fg of G. citricarpa and G. mangiferae DNA was possible. Pycnidiospore disruption resulted in detection of single pycnidiospores with 78 (59 to 102; 95% confidence interval [CI]) and 112 (92 to 136; 95% CI) ITS copies for G. citricarpa and G. mangiferae, respectively. Detection was from partially decomposed leaves where fruiting bodies cannot be morphologically distinguished. Temperature and wetting period have significant effects on Guignardia spp. pseudothecia production in leaf litter. Based on relative biomass or the proportion of nuclei detected, G. citricarpa and G. mangiferae respond more strongly to wetting period than temperature. This qPCR assay will provide additional epidemiological data on black spot in tissues where G. citricarpa and G. mangiferae are not easily distinguished.

Multifunctional Surface, Subsurface, and Systemic Therapeutic (MS3T) Formulation for the Control of Citrus Canker.

A multifunctional surface, subsurface and systemic therapeutic (MS3T) formulation comprised of two bactericides, both didecyldimethylammonium chloride (DDAC) and a zinc (Zn)-chelate, was developed as an alternative to copper pesticides for crop protection. Agricultural grade chemicals were used to prepare MS3T formulations. Minimal inhibitory concentration (MIC) was determined to be tested in vitro against Xanthomonas alfalfae subsp. citrumelonis (herein called Xa), Escherichia coli (E. coli), and Pseudomonas syringae (Ps). Assessment of the phytotoxic potential was carried out on tomato under greenhouse conditions. Moreover, field trials were conducted during three consecutive years on grapefruit (Chrysopelea paradise) groves to evaluate efficacy against citrus canker (Xanthomonas citri subsp. citri), scab (Elsinoe fawcetti), and melanose (Diaporthe citri). In addition to disease control, improvements to both fruit yield and quality were observed likely due to the nutritional activity of MS3T via the sustained release of plant nutrients (Zn and nitrogen). Zn residues of leaf tissues were analyzed via atomic absorption spectroscopy (AAS) at various time points before and after MS3T foliar applications throughout the duration of the 2018 field trial. Field trial results demonstrated MS3T to be an effective alternative to copper (Cu)-based formulations for the control of citrus canker.

4-Nitrotryptophan is a substrate for the non-ribosomal peptide synthetase TxtB in the thaxtomin A biosynthetic pathway.

Thaxtomin A, a cyclic dipeptide with a nitrated tryptophan moiety, is a phytotoxic pathogenicity determinant in scab-causing Streptomyces species that inhibits cellulose synthesis by an unknown mechanism. Thaxtomin A is produced by the action of two non-ribosomal peptide synthetase modules (TxtA and TxtB) and a complement of modifying enzymes, although the order of biosynthesis has not yet been determined. Analysis of a thaxtomin dual module knockout mutant and single module knockout mutants revealed that 4-nitrotryptophan is an intermediate in thaxtomin A biosynthesis prior to backbone assembly. The 4-nitrotryptophan represents a novel substrate for non-ribosomal peptide synthetases. Through identification of N-methyl-4-nitrotryptophan in a single module knockout and the use of adenylation domain specificity prediction software, TxtB was identified as the non-ribosomal peptide synthetase module specific for 4-nitrotryptophan.

Plant-pathogenic Streptomyces species produce nitric oxide synthase-derived nitric oxide in response to host signals.

Nitric oxide (NO) is a potent intercellular signal for defense, development, and metabolism in animals and plants. In mammals, highly regulated nitric oxide synthases (NOSs) generate NO. NOS homologs exist in some prokaryotes, but direct evidence for NO production by these proteins has been lacking. Here, we demonstrate that a NOS in plant-pathogenic Streptomyces species produces diffusible NO. NOS-dependent NO production increased in response to cellobiose, a plant cell wall component, and occurred at the host-pathogen interface, demonstrating induction by host signals. These data document in vivo production of NO by prokaryotic NOSs and implicate pathogen-derived NO in host-pathogen interactions. NO may serve as a signaling molecule in other NOS-containing bacteria, including the medically and environmentally important organisms Bacillus anthracis, Staphylococcus aureus, and Deinococcus radiodurans.

Control of Citrus Canker in Greenhouse and Field with a Zinc, Urea, and Peroxide Ternary Solution.

Accumulation of toxic copper in soil and development of copper-resistant pests are emerging challenges currently faced by the agricultural community worldwide. As an alternative, we have developed a ternary zinc chelate solution (TSOL) pesticide where zinc ions are the primary active ingredient. The material is composed of zinc, urea, and hydrogen peroxide. Urea was chosen as it is widely used as a plant fertilizer and can also bind to both zinc and hydrogen peroxide. No phytotoxicity was observed with TSOL on Meyer lemon (Citrus × meyeri) seedlings at a field spray rate of 800 µg/mL Zn metal concentration. Antimicrobial studies showed that TSOL exhibited improved killing efficacy against Escherichia coli and Xanthomonas alfalfae compared to Zn ions alone. Citrus canker field trials in a grapefruit (Chrysopelea paradisi) grove over three years showed that TSOL provided comparable disease protection to copper products at an equivalent or lower metal content.

Multimodal Generally Recognized as Safe ZnO/Nanocopper Composite: A Novel Antimicrobial Material for the Management of Citrus Phytopathogens.

Copper (Cu) bactericides/fungicides are used extensively for crop protection in agriculture. Concerns for Cu accumulation in soil, Cu leaching into the surrounding ecosystem, and development of Cu resistance in phytopathogenic bacteria are evident. While there is no suitable alternative to Cu available to date for agricultural uses, it is possible to reduce Cu per application by supplementing with Zn and improving Cu bioavailability using nanotechnology. We have prepared a non-phytotoxic composite material consisting of generally recognized as safe ZnO 800 particles and nanocopper-loaded silica gel (ZnO-nCuSi). The morphology of the ZnO-nCuSi material was characterized using scanning electron microscopy, showing ZnO particles dispersed in the silica gel matrix. ZnO-nCuSi demonstrated strong in vitro antimicrobial properties against several model plant bacterial species. Two consecutive year field efficacy results showed that agri-grade ZnO-nCuSi was effective in controlling citrus canker disease at less than half the metallic rate of the commercial cuprous oxide/zinc oxide pesticide.

Use of real-time PCR to discriminate parasitic and saprophagous behaviour by nematophagous fungi.

Entomopathogenic nematodes (EPNs) are important pathogens of soilborne insects and are sometimes developed commercially to manage insect pests. Numerous nematophagous fungal species (NF) prey on nematodes and are thought to be important in regulating natural or introduced EPN populations. However, nematophagy by these fungi in nature cannot be inferred using existing methods to estimate their abundance in soil because many of these fungi are saprophytes, resorting to parasitism primarily when certain nutrients are limiting. Therefore, we developed an assay to quantify NF DNA in samples of nematodes. Species-specific primers and TaqMan probes were designed from the ITS rDNA regions of Arthrobotrys dactyloides, Arthrobotrys oligospora, Arthrobotrys musiformis, Gamsylella gephyropagum and Catenaria sp. When tested against 23 non-target fungi, the TaqMan real-time PCR assay provided sensitive and target-specific quantification over a linear range. The amount of A. dactyloides or Catenaria sp. DNA in 20 infected nematodes, measured by real-time PCR, differed between fungal species (P=0.001), but not between experiments (P>0.05). However, estimates of relative NF parasitism using a bioassay with 20 nematodes infected by either species, differed greatly (P<0.001) depending on whether the fungi were alone or combined in the samples used in the assay. Tests done to simulate detection of NF DNA in environmental samples showed that, for all species, background genomic DNA and/or soil contaminants reduced the quantity of DNA detected. Nested PCR was ineffective for increasing the detection of NF in environmental samples. Indeed, real-time PCR detected higher amounts of NF DNA than did nested PCR. The spatial patterns of NF parasitism in a citrus orchard were derived using real-time PCR and samples of nematodes extracted from soil. The parasitism by Catenaria sp. was positively related to the abundance of both heterorhabditid and steinernematid EPNs. The possible significance of the associations is ambiguous because NF attack a broad range of nematode taxa whereas EPNs are a small minority of the total nematode population in a soil sample. These studies demonstrate the potential of real-time PCR to study the role of NF parasitism in soil food webs.

Thaxtomin biosynthesis: the path to plant pathogenicity in the genus Streptomyces.

Streptomyces species are best known for their ability to produce a wide array of medically and agriculturally important secondary metabolites. However, there is a growing number of species which, like Streptomyces scabies, can function as plant pathogens and cause scab disease on economically important crops such as potato. All of these species produce the phytotoxin thaxtomin, a nitrated dipeptide which inhibits cellulose synthesis in expanding plant tissue. The biosynthesis of thaxtomin involves conserved non-ribosomal peptide synthetases, P450 monooxygenases, and a nitric oxide synthase, the latter being required for nitration of the toxin. This nitric oxide synthase is also responsible for the production of diffusible nitric oxide by scab-causing streptomycetes at the host-pathogen interface, suggesting that nitric oxide production might play an additional role during the infection process. The thaxtomin biosynthetic genes are transcriptionally regulated by an AraC/XylS family regulator, TxtR, which is conserved in pathogenic streptomycetes and is encoded within the thaxtomin biosynthetic gene cluster. The TxtR protein specifically binds cellobiose, a known inducer of thaxtomin biosynthesis, and cellobiose is required for expression of the biosynthetic genes. A second virulence gene in pathogenic Streptomyces species, nec1, encodes a novel secreted protein that may suppress plant defence responses. The thaxtomin biosynthetic genes and nec1 are contained on a large mobilizable pathogenicity island; the transfer of this island to recipient streptomycetes likely explains the rapid emergence of new pathogenic species. The newly available genome sequence of S. scabies will provide further insight into the mechanisms utilized by pathogenic streptomycetes during plant-microbe interactions.

The AraC/XylS regulator TxtR modulates thaxtomin biosynthesis and virulence in Streptomyces scabies.

Streptomyces scabies is the best studied of those streptomycetes that cause an economically important disease known as potato scab. The phytotoxin thaxtomin is made exclusively by these pathogens and is required for virulence. Here we describe regulation of thaxtomin biosynthesis by TxtR, a member of the AraC/XylS family of transcriptional regulators. The txtR gene is imbedded in the thaxtomin biosynthetic pathway and is located on a conserved pathogenicity island in S. scabies, S. turgidiscabies and S. acidiscabies. Thaxtomin biosynthesis was abolished and virulence was almost eliminated in the txtR deletion mutant of S. scabies 87.22. Accumulation of thaxtomin biosynthetic gene (txtA, txtB, txtC, nos) transcripts was reduced compared with the wild-type S. scabies 87.22. NOS-dependent nitric oxide production by S. scabies was also reduced in the mutant. The TxtR protein bound cellobiose, an inducer of thaxtomin production, and transcription of txtR and thaxtomin biosynthetic genes was upregulated in response to cellobiose. TxtR is the first example of an AraC/XylS family protein regulated by cellobiose. Together, these data suggest that cellobiose, the smallest oligomer of cellulose, may signal the availability of expanding plant tissue, which is the site of action of thaxtomin.