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Fungus farming ambrosia beetles carry their nutritional mutualistic fungi in specialized structures called mycetangia. Fungal propagules are also dispersed phoretically on the beetle’s exoskeleton. We determined the phoretic presence and abundance of Raffaelea lauricola, the causal agent of the laurel wilt disease in avocado, on five ambrosia beetle species: Xyleborus bispinatus, Xyleborus volvulus, Xyleborus affinis, Xyleborinus saxesenii and Xylosandrus crassiusculus. Beetles were captured while in flight, excavated from logs, and from logs placed in emergence chambers. Beetles collected by the three methods were assayed for the presence of internal (gut and mycetangium) and external (attached to the exoskeleton) colony forming units (CFUs) of R. lauricola. The pathogen was recovered from the exoskeleton of all beetle species. The collection method significantly influenced the frequency of pathogen recovery, and the abundance of both internal and phoretic R. lauricola was species-specific. Internal CFUs recovery was greater than phoretic recovery. Besides R. lauricola, other cycloheximide tolerant fungi, including mutualistic and entomopathogenic fungi, were isolated from the beetle’s exoskeleton. However, phoretic CFUs of R. lauricola were more prevalent and abundant than any other mutualistic phoretic fungi across the beetle species. Our results suggest that phoresy is a common mechanism of transportation of wood-inhabiting fungi and that phoretic transmission of R. lauricola may potentially contribute to the infection in avocado.

The bacterial and fungal community involved in ambrosia beetle fungiculture remains poorly studied compared to the famous fungus-farming ants and termites. Here we studied microbial community dynamics of laboratory nests, adults, and brood during the life cycle of the sugarcane shot hole borer, Xyleborus affinis. We identified a total of 40 fungal and 428 bacterial operational taxonomic units (OTUs), from which only five fungi (a Raffaelea fungus and four ascomycete yeasts) and four bacterial genera (Stenotrophomonas, Enterobacter, Burkholderia, and Ochrobactrum) can be considered the core community playing the most relevant symbiotic role. Both the fungal and bacterial populations varied significantly during the beetle’s life cycle. While the ascomycete yeasts were the main colonizers of the gallery early on, the Raffaelea and other filamentous fungi appeared after day 10, at the time when larval hatching happened. Regarding bacteria, Stenotrophomonas and Enterobacter dominated overall but decreased in foundresses and brood with age. Finally, inferred analyses of the putative metabolic capabilities of the bacterial microbiome revealed that they are involved in (i) degradation of fungal and plant polymers, (ii) fixation of atmospheric nitrogen, and (iii) essential amino acid, cofactor, and vitamin provisioning. Overall, our results suggest that yeasts and bacteria are more strongly involved in supporting the beetle-fungus farming symbiosis than previously thought., Research reported in this publication was supported by CONACyT-FORDECYT number 292399, Spanish Ministry of Economy and Competitiveness under award numbers SAF2015-65878-R and PGC2018-099344-B-I00, cofinanced by the European Regional Development Fund (ERDF), and from Generalitat Valenciana (project Prometeo/2018/A133). P.H.W.B. was supported by the German Research Foundation (DFG Emmy Noether grant BI 1956/1-1).

Survival of ambrosia beetles relies on obligate nutritional relationships with fungal symbionts that are cultivated in tunnels excavated in the sapwood of their host trees. The dynamics of fungal associates, along with the developmental biology, and gallery construction of the ambrosia beetle Xyleborus bispinatus were elaborated. One generation of this ambrosia beetle was reared in an artificial medium containing avocado sawdust. The developmental time from egg to adult ranged from 22 to 24 d. The mean total gallery length (14.4 cm and 13 tunnels) positively correlated with the number of adults. The most prevalent fungal associates were Raffaelea arxii in the foundress mycangia and new galleries, and Raffaelea subfusca in the mycangia of the F1 adults and the final stages of the galleries. Raffaelea sp. PL1001, Raffaelea subalba and seven yeast species were also recovered. These results indicate flexibility in the association between X. bispinatus and its symbiont species, and that this beetle may use more than one Raffaelea species as a food source. These results are important, as X. bispinatus has been associated with the transmission of Raffaelea lauricola, the causal agent of laurel wilt, a lethal disease affecting avocado trees.

Ambrosia beetles in the Euwallacea nr. fornicatus complex (Coleoptera: Curculionidae) vector Fusarium spp. fungi pathogenic to susceptible hosts, including avocado, Persea americana Mill., (Lauraceae). Previous survey traps in Florida avocado groves indicated significant beetle populations in several groves with minimal observed beetle activity, suggesting an external beetle source. A natural area near one such grove revealed E. nr. Fornicates colonization of wild tamarind, Lysiloma latisiliquum (L.) Bentham (Fabaceae). A survey of the natural area was conducted to understand the role that natural areas might play in E . nr. fornicatus ecology in southern Florida. Headspace volatiles from rasped avocado and L. latisiliquum bark were analyzed by gas chromatography-mass spectroscopy (GC-MS) to identify potential attractants. Genetic analysis confirmed that these beetles and their symbiotic fungi are of the same complex that attacks Florida avocado. Gas chromatography-mass spectroscopy analysis indicated that avocado is high in α-copaene (a known attractant of E . nr. fornicatus ), but this kairomone is lacking in L. latisiliquum . Host diam and ht were examined for potential influence on colonization behavior. Albizia lebbeck (L.) Bentham (Fabaceae) and an unknown shrub also were observed to be suitable hosts. Concurrent with this study, a nearby grove of soursop, Annona muricata L. (Annonaceae), was found to have infestations of E. nr. fornicatus . Euwallacea nr. fornicatus populations are increasing in Florida and other cultivated and native trees are potentially at risk. Further research is warranted to better understand the ecology of this emerging pest and the chemical cues used for host location. Resumen Los escarabajos ambrosiales en el complejo Euwallacea cr. fornicatus (Coleoptera: Curculionidae) transmiten hongos patogenos de Fusarium spp. a hospederos susceptibles, incluyendo el aguacate, Persea americana Mill., (Lauraceae). Trampas usadas anteriormente para monitorear los huertos de aguacate en la Florida indicaron poblaciones significativas de escarabajos en varios huertos con una actividad minima observada sobre los arboles de aguacate lo que sugiere una fuente externa de escarabajos. Un area natural cerca de uno de estos huertos revelo la colonizacion de E. cr. Fornicates sobre el tamarindo silvestre, Lysiloma latisiliquum (L.) Bentham (Fabaceae). Se realizo un sondeo del area natural para comprender el papel que las areas naturales podrian jugar en la ecologia de E. cr. fornicatus en el sur de la Florida. Se analizaron los volatiles “headspace” del aguacate raspado y la corteza de L. latisiliquum por medio del cromatografia de gases y espectrometria de masas (CG-SM) para identificar atrayentes potenciales. El analisis genetico confirmo que estos escarabajos y sus hongos simbioticos son del mismo complejo que ataca al aguacate de Florida. El analisis de cromatografia de gases y espectrometria de masas indico que el aguacate tiene un alto contenido de α-copaeno (un atrayente conocido de E. cr. fornicatus ), pero hace falta esta kairomona en L. latisiliquum . Se examinaron el diametro y la altura del hospedero para determinar su posible influencia sobre el comportamiento de colonizacion. Tambien, se observaron Albizia lebbeck (L.) Bentham (Fabaceae) y un arbusto desconocido como hospederos adecuados. Simultaneamente con este estudio, se encontro que un huerto de guanabana ( Annona muricata L.: Annonaceae) cercano, que tenia infestaciones de E . cr. fornicatus . Las poblaciones de Euwallacea cr. fornicatus estan incrementando en la Florida y otros arboles cultivados y nativos estan potencialmente en riesgo. Se necesita mas investigacion para comprender mejor la ecologia de esta plaga emergente y las senales quimicas que utiliza para localizar los hospederos. View this article in BioOne

In the present study, we report the in-vitro antifungal activity of ZnO nanoparticles (NPs) against three species of the ambrosia beetle mutualistic fungi, Raffaelea arxii, Raffaelea subfusca, Raffaelea lauricola. The latter is the causal agent of laurel wilt, a deadly vascular disease of avocado. The effect of coating and size of two kinds of ZnO-NPs on the antifungal activity against those mutualistic is presented. The NPs obtained by the hydrothermal method (hyd-ZnO) were uncoated plate-like NPs with sizes between 200 and 600 nm, while the NPs obtained by sol–gel (sg-ZnO) were PEG-coated semi-spherical NPs with ~60 nm in size. Although the inhibition percentage was higher than 80% for both kinds of NPs (at a concentration of 0.5 mg/ml), the highest antifungal capacity was exhibited by the hyd-ZnO-NPs. Here we show that despite the size of the NPs, which is a crucial parameter, the coating of ZnO-NPs is the most critical factor for the improvement of their antifungal properties.

MopB is a major outer membrane protein (OMP) in Xylella fastidiosa, a bacterial plant pathogen that causes losses on many economically important crops. Based on in silico analysis, the uncharacterized MopB protein of X. fastidiosa contains a β-barrel structure with an OmpA-like domain and a predicted calcium-binding motif. Here, MopB function was studied by mutational analysis taking advantage of the natural competence of X. fastidiosa. Mutants of mopB were constructed in two different X. fastidiosa strains, the type strain Temecula and the more virulent WM1-1. Deletion of the mopB gene impaired cell-to-cell aggregation, surface attachment, and biofilm formation in both strains. Interestingly, mopB deletion completely abolished twitching motility. Electron microscopy of the bacterial cell surface revealed that mopB deletion eliminated type IV and type I pili formation, potentially caused by destabilization of the outer membrane. Both mopB mutants showed reduced virulence using tobacco (Nicotiana tabacum) as a host under greenhouse conditions. These results suggest that MopB has pleiotropic functions in biofilm formation and twitching motility and is important for virulence of X. fastidiosa.

Like other ambrosia beetles, Xyleborus volvulus Fabricius (Coleoptera: Curculionidae) lives in a mutualistic symbiotic relationship with fungi that serve as food source. Until recently, X. volvulus was not considered a pest, and none of its symbionts were considered plant pathogens. However, recent reports of an association between X. volvulus and Raffaelea lauricola T.C. Harr., Fraedrich & Aghayeva (Ophiostomatales: Ophiostomataceae), the cause of the laurel wilt disease of avocado (Persea americana Mill. [Laurales: Lauraceae]), and its potential role as vector of the pathogen merit further investigation. The objective of this study was to evaluate three artificial media containing sawdust obtained from avocado or silkbay (Persea humilis Nash) for laboratory rearing of X. volvulus. The effect of R. lauricola in the media on the beetle's reproduction was also evaluated. Of the three media, the one with the lowest content of sawdust and intermediate water content provided the best conditions for rearing X. volvulus. Reproduction on this medium was not affected by the sawdust species or the presence of R. lauricola. On the other two media, there was a significant interaction between sawdust species and R. lauricola. The presence of R. lauricola generally had a negative effect on brood production. There was limited colonization of the mycangia of X. volvulus by R. lauricola on media inoculated with the pathogen. From galleries formed within the best medium, there was 50% recovery of R. lauricola, but recovery was much less from the other two media. Here, we report the best artificial substrate currently known for X. volvulus.

The ambrosia beetle Xyleborus volvulus Fabricius has been reported as a potential vector of the plant pathogen Raffaelea lauricola T.C. Harr., Fraedrich & Aghayeva that is affecting avocado orchards in South Florida. In this study, we examined its life cycle, process of gallery formation, gallery structure, and fungal associates by rearing one generation on avocado sawdust medium under control conditions. The adult foundress excavated a vertical tunnel that constituted the main gallery with a length of 2.5 cm, followed by the construction of up to six secondary galleries with a total length of 4.4 cm. The time period for one generation (egg to adult) was 28 days. Teneral males emerged 3 days after the emergence of the first females. The F(1) generation did not significantly contribute to gallery expansion. Four species of Raffaelea and nine yeast species were recovered from galleries and beetles. Raffaelea arxii and Candida berthetii were the most frequent symbionts recovered from new adults and galleries. Candida berthetii dominated during the early stages of the gallery development, whereas R. arxii was most frequent in later stages. Other Raffaelea species were inconsistently isolated from galleries, which suggests a strong association between Xyleborus volvulus and both R. arxii and C. berthetii. These results suggest that R. arxii is the primary nutritional symbiont of X. volvulus and that yeast species may be pioneer colonizers that assist with the growth of fungal symbionts. IMPORTANCE Ambrosia beetles cultivate fungi in tunnels bored into weakened host trees. This obligate interaction is required for their survival as beetles feed on these symbiotic fungi, and the fungi benefit from transportation by the beetles. Xyleborus volvulus carries many nonpathogenic symbionts; however, recently the acquisition of Raffaelea lauricola (the causal agent of a lethal vascular disease of lauraceous trees) by this beetle has altered its status from wood degrader to potential pest in avocado. We conducted a study to understand the relationship of this beetle and its fungal associates. Our results show that X. volvulus has a multipartite flexible association with different Raffaelea species. The lack of fidelity in the mutualistic association may explain the acquisition of R. lauricola. Knowing the beetle biology and its mutualistic interactions furthers an understanding of the beetle’s role as a potential vector and in disease transmission.

The tea shot-hole borer (THSB), a member of the Euwallacea fornicatus (Eichhoff 1868) complex of species, harboring the plant pathogenic symbiotic fungi (AF-6, AF-8, and Fusarium sp.) (Curculionidae: Scolytinae) attacks avocado (Persea americana Miller) and other tree species in Florida. Currently, their management is limited to prevention and sanitation; therefore, the development of novel control alternatives is critical to mitigate pest outbreaks and avoid massive crop losses. Here, we report on the antifungal activity of copper nanoparticles (Cu-NPs) against the beetle/fungus complex and their effect on the beetle's reproduction. The results revealed that the synthesized Cu-NPs were pure metallic Cu with an average size of 240.88 ± 62.66 nm measured by electron microscopy. The antifungal test for symbionts showed that 0.75 mg/mL of Cu-NPs caused growth inhibition above 60%. However, strong alterations in the morphology of fungal cells were observed at Cu-NPs concentrations as low as 0.1 mg/mL. Additionally, treatments with 0.25, 0.5, and 1 mg/mL of Cu-NPs led to a reduction in offspring production in a concentration dependent manner. According to our findings, the application of Cu-NPs could be potentially considered as an alternative for the control of the beetle-fungi complex or even be integrated into novel disease management strategies.

Triticum mosaic virus (TriMV) was discovered in 2006 and affects wheat (Triticum spp.) production systems in the Great Plains of the United States. Genetic resistance is available in a few commercial varieties; however, it is not effective above 20°C. RNA interference (RNAi) was evaluated as an alternative strategy to generate resistance to TriMV. An RNAi pANDA-mini-based hairpin expression vector was created from 272 bp of the TriMV coat protein (CP) sequence. Immature embryos of the wheat cultivar ‘Bobwhite’ were co-transformed by biolistic particle delivery system with the pANDA::TriMVCP vector and pAHC20, containing the bar gene for glufosinate selection. T₀ plants were analyzed by PCR for the presence of the RNAi TriMV CP fragment. Transgenic T₁ lines were tested for transgene expression via reverse transcription PCR Virus resistance was evaluated by mechanical inoculation with TriMV and indirect enzyme-linked immunosorbant assay (ELISA). Several lines were identified that were resistant, having little or no viral RNA. Resistant lines underwent single plant selection to the T₆ generation. Real-time PCR and ELISA showed that T₆ lines had a high level of resistance when challenged with the virus. To test if the transgene would behave similarly in an adapted variety, homozygous transgenic T₃ lines were crossed with the virus-susceptible winter wheat, ‘Overley.’ Real-time PCR results indicate decreased viral titer by as much as 20-fold in the T₆ transgenic lines, the F₁ crosses, and the BC₁F₁ lines when compared with control plants, providing evidence that this RNAi-silencing construct can provide stable resistance to TriMV and has great potential benefits to both breeders and producers.

The lychee erinose mite (LEM), Aceria litchii (Keifer) is a serious pest of lychee (Litchi chinensis Sonn.). LEM causes a type of gall called &lsquo, erineum&rsquo, (abnormal felty growth of trichomes from the epidermis), where it feeds, reproduces and protects itself from biotic and abiotic adversities. In February of 2018, LEM was found in a commercial lychee orchard on Pine Island, Florida. Infestations were recorded on young leaves, stems, and inflorescences of approximately 30 young trees (1.5&ndash, 3.0 yrs.) of three lychee varieties presenting abundant new growth. Although LEM is present in Hawaii, this mite is a prioritized quarantine pest in the continental USA and its territories. Florida LEM specimens showed small morphological differences from the original taxonomic descriptions of Keifer (1943) and Huang (2008). The observed differences are probably an artifact of the drawings in the original descriptions. Molecular comparisons were conducted on the DNA of LEM specimens from India, Hawaii, Brazil, Taiwan, Australia and Florida. The amplified COI fragment showed very low nucleotide variation among the locations and thus, could be used for accurate LEM identification. The ITS1 sequences and partial 5.8S fragments displayed no nucleotide differences for specimens from any of the locations except Australia. Consistent differences were observed in the ITS2 and 28S fragments. The ITS1-ITS2 concatenated phylogeny yielded two lineages, with Australia in one group and Hawaii, India, Brazil, Florida and Taiwan in another. Specimens from Taiwan and Florida present identical ITS and rDNA segments, suggesting a common origin, however, analysis of additional sequences is needed to confirm the origin of the Florida population.

The plant-pathogenic bacterium Xylella fastidiosa is restricted to the xylem vessel environment, where mineral nutrients are transported through the plant host; therefore, changes in the concentrations of these elements likely impact the growth and virulence of this bacterium. Twitching motility, dependent on type IV pili (TFP), is required for movement against the transpiration stream that results in basipetal colonization. We previously demonstrated that calcium (Ca) increases the motility of X. fastidiosa , although the mechanism was unknown. PilY1 is a TFP structural protein recently shown to bind Ca and to regulate twitching and adhesion in bacterial pathogens of humans. Sequence analysis identified three pilY1 homologs in X. fastidiosa (PD0023, PD0502, and PD1611), one of which (PD1611) contains a Ca-binding motif. Separate deletions of PD0023 and PD1611 resulted in mutants that still showed twitching motility and were not impaired in attachment or biofilm formation. However, the response of increased twitching at higher Ca concentrations was lost in the pilY1 -1611 mutant. Ca does not modulate the expression of any of the X. fastidiosa PilY1 homologs, although it increases the expression of the retraction ATPase pilT during active movement. The evidence presented here suggests functional differences between the PilY1 homologs, which may provide X. fastidiosa with an adaptive advantage in environments with high Ca concentrations, such as xylem sap.

Wheat streak mosaic virus (WSMV) is one of the major wheat viruses found in the Great Plains of the USA. Cultural practices are the primary methods of disease management, though not fully effective. Although genetic resistance is available, it is temperature sensitive and is sometimes closely linked with traits having negative agronomic effects. Alternative approaches to viral resistance are clearly needed. RNA interference (RNAi) has been shown to play a role in viral defense response and has been successfully used as a biotechnological tool to preprogram viral resistance in transgenic plants. In this work, a portion of the coat protein of WSMV was used as a hairpin construct and was co-transformed with pAHC20-bar to elicit viral resistance. Eleven WSMV RNAi independent transgenic events were obtained. Thirteen T1 lines were resistant as evident by the lack of viral RNA within the tissue. Beginning in the T2 generation, single-plant lineages were selected, selfed, and evaluated for resistance and presence of the transgene until the T5 generation. Families were then evaluated for the presence of the transgene, presence of the selectable marker, and WSMV resistance. Each of the lines in the T5 generation were resistant to the virus. Generational selection has maintained expression of the transgene and resistance to WSMV.

Members of a complex of cryptic species, that correspond morphologically to the ambrosia beetle Euwallacea fornicatus (Eichhoff) (Coleoptera: Curculionidae: Scolytinae), were recently found attacking avocado (Persea americana Mill.) in Israel and California. In early 2016, an outbreak of another member of this species complex was detected infesting approximately 1500 avocado trees in an avocado orchard at Homestead, Florida. An area-wide survey was conducted in commercial avocado groves of Miami-Dade County, Florida to determine the distribution and abundance of E. nr. fornicatus, to identify different populations of E. nr. fornicatus and their fungal associates, and to assess the extent of damage to avocado trees. Ewallacea nr. fornicatus were captured in 31 of the 33 sampled sites. A sample of 35 beetles from six different locations was identified as E. nr. fornicatus sp. #2, which is genetically distinct from the species causing damage in California and Israel. Eleven fungal associates were identified: an unknown Fusarium sp., AF-8, AF-6, Graphium euwallaceae, Acremonium sp. Acremonium morum, Acremonium masseei, Elaphocordyceps sp. and three yeast species. The unknown Fusarium isolates were the most abundant and frequently found fungus species associated with adult beetles and lesions surrounding the beetle galleries. In addition to fungal associates, three bacteria species were found associated with adult E. nr. fornicatus. Visual inspections detected significant damage in only two orchards. A large number of beetles were captured in locations with no apparent damage on the avocado trees suggesting that E. nr. fornicatus are associated with other host(s) outside the groves or with dead trees or branches inside the groves. More research is needed to determine the potential threat E. nr. fornicatus and its fungal associates pose to the avocado industry and agricultural and natural ecosystems in Florida.

Like other members of the tribe Xyleborini, Xyleborus bispinatus Eichhoff can cause economic damage in the Neotropics. X. bispinatus has been found to acquire the laurel wilt pathogen Raffaelea lauricola (T. C. Harr., Fraedrich & Aghayeva) when breeding in a host affected by the pathogen. Its role as a potential vector of R. lauricola is under investigation. The main objective of this study was to evaluate three artificial media, containing sawdust of avocado (Persea americana Mill.) and silkbay (Persea humilis Nash.), for rearing X. bispinatus under laboratory conditions. In addition, the media were inoculated with R. lauricola to evaluate its effect on the biology of X. bispinatus. There was a significant interaction between sawdust species and R. lauricola for all media. Two of the media supported the prolific reproduction of X. bispinatus, but the avocado-based medium was generally more effective than the silkbay-based medium, regardless whether or not it was inoculated with R. lauricola. R. lauricola had a neutral or positive effect on beetle reproduction. The pathogen was frequently recovered from beetle galleries, but only from a few individuals which were reared on inoculated media, and showed limited colonization of the beetle’s mycangia. Two media with lower water content were most effective for rearing X. bispinatus.

Twitching motility, involving type IV pili, is essential for host colonization and virulence of many pathogenic bacteria. Studies of PilY1, a tip-associated type IV pili protein, indicate that PilY1 functions as a switch between pilus extension and retraction, resulting in twitching motility. Recent work detected a calcium-binding motif in PilY1 of some animal bacterial pathogens and demonstrated that binding of calcium to PilY1 with this motif regulates twitching. Though studies of PilY1 in non-animal pathogens are limited, our group demonstrated that twitching motility in the plant pathogen Xylella fastidiosa , which contains three PilY1 homologs, is increased by calcium supplementation. A study was conducted to investigate the phylogenetic relationship between multiple PilY1 homologs, the presence of calcium-binding motifs therein, and calcium-mediated twitching motility across diverse bacteria. Strains analyzed contained one to three PilY1 homologs, but phylogenetic analyses indicated that PilY1 homologs containing the calcium-binding motif Dx[DN]xDGxxD are phylogenetically divergent from other PilY1 homologs. Plant-associated bacteria included in these analyses were then examined for a calcium-mediated twitching response. Results indicate that bacteria must have at least one PilY1 homolog containing the Dx[DN]xDGxxD motif to display a calcium-mediated increase in twitching motility, which likely reflects an adaption to environmental calcium concentrations.

Summary The 2 cryptic species of Euwallacea nr. fornicatus (Coleoptera: Curculionidae: Scolytinae) have more than 300 known hosts, of which avocado is the most important. Fusarium dieback, the fungal disease associated with these beetles, has killed some host plant species in Israel and the USA. Kuroshio shot hole borer, one of the 2 cryptic species, is reported for the first time in Baja California, Mexico, and represents a new threat to avocado and other potential hosts in urban and natural areas.

Xylella fastidiosa is a bacterial plant pathogen that infects numerous plant hosts. Disease develops when the bacterium colonizes the xylem vessels and forms a biofilm. Inductively coupled plasma optical emission spectroscopy was used to examine the mineral element content of this pathogen in biofilm and planktonic states. Significant accumulations of copper (30-fold), manganese (6-fold), zinc (5-fold), calcium (2-fold) and potassium (2-fold) in the biofilm compared to planktonic cells were observed. Other mineral elements such as sodium, magnesium and iron did not significantly differ between biofilm and planktonic cells. The distribution of mineral elements in the planktonic cells loosely mirrors the media composition; however the unique mineral element distribution in biofilm suggests specific mechanisms of accumulation from the media. A cell-to-surface attachment assay shows that addition of 50 to 100 µM Cu to standard X. fastidiosa media increases biofilm, while higher concentrations (>200 µM) slow cell growth and prevent biofilm formation. Moreover cell-to-surface attachment was blocked by specific chelation of copper. Growth of X. fastidiosa in microfluidic chambers under flow conditions showed that addition of 50 µM Cu to the media accelerated attachment and aggregation, while 400 µM prevented this process. Supplementation of standard media with Mn showed increased biofilm formation and cell-to-cell attachment. In contrast, while the biofilm accumulated Zn, supplementation to the media with this element caused inhibited growth of planktonic cells and impaired biofilm formation. Collectively these data suggest roles for these minerals in attachment and biofilm formation and therefore the virulence of this pathogen.

Xylella fastidiosa is a plant-pathogenic bacterium that forms biofilms inside xylem vessels, a process thought to be influenced by the chemical composition of xylem sap. In this work, the effect of calcium on the production of X. fastidiosa biofilm and movement was analyzed under in vitro conditions. After a dose-response study with 96-well plates using eight metals, the strongest increase of biofilm formation was observed when medium was supplemented with at least 1.0 mM CaCl 2 . The removal of Ca by extracellular (EGTA, 1.5 mM) and intracellular [1,2-bis( o -aminophenoxy)ethane- N , N , N ′, N ′-tetraacetic acid acetoxymethyl ester (BAPTA/AM), 75 μM] chelators reduced biofilm formation without compromising planktonic growth. The concentration of Ca influenced the force of adhesion to the substrate, biofilm thickness, cell-to-cell aggregation, and twitching motility, as shown by assays with microfluidic chambers and other assays. The effect of Ca on attachment was lost when cells were treated with tetracycline, suggesting that Ca has a metabolic or regulatory role in cell adhesion. A double mutant ( fimA pilO ) lacking type I and type IV pili did not improve biofilm formation or attachment when Ca was added to the medium, while single mutants of type I ( fimA ) or type IV ( pilB ) pili formed more biofilm under conditions of higher Ca concentrations. The concentration of Ca in the medium did not significantly influence the levels of exopolysaccharide produced. Our findings indicate that the role of Ca in biofilm formation may be related to the initial surface and cell-to-cell attachment and colonization stages of biofilm establishment, which rely on critical functions by fimbrial structures.