First Report of HLB Causal Agent in Psyllid in State of Bahia, Brazil.

The state of Bahia ranks fourth in the national rank for citrus production, and the region of Chapada Diamantina is emerging an important producer of orange for fresh fruit market. Huanglongbing (HLB) is the major phytosanitary threat to Bahia citriculture. In Brazil, the disease was first reported in 2004 in São Paulo state. The bacterium Candidatus Liberibacter asiaticus (CLas) is one of the causal agents of HLB, which is transmitted by the insect vector Diaphorina citri Kuwayama (Hemiptera: Liviidae). Bahia is a HLB-free area; therefore, it is essential to monitor its citrus-producing areas to early detect any possible introduction of the CLas. This study aimed to monitor the presence of the bacteria in the insect vector. Diaphorina citri samples were collected from 2011 to 2014 in different cities located at Chapada Diamantina region and tested by qPCR for the presence of CLas. Three samples were considered positive to bacterium, and all from psyllids collected on Murraya paniculata in the city of Seabra.

Diversity and phenotypic analyses of salt- and heat-tolerant wild bean Phaseolus filiformis rhizobia native of a sand beach in Baja California and description of Ensifer aridi sp. nov.

In northern Mexico, aridity, salinity and high temperatures limit areas that can be cultivated. To investigate the nature of nitrogen-fixing symbionts of Phaseolus filiformis, an adapted wild bean species native to this region, their phylogenies were inferred by MLSA. Most rhizobia recovered belong to the proposed new species Ensifer aridi. Phylogenetic analyses of nodC and nifH show that Mexican isolates carry symbiotic genes acquired through horizontal gene transfer that are divergent from those previously characterized among bean symbionts. These strains are salt tolerant, able to grow in alkaline conditions, high temperatures, and capable of utilizing a wide range of carbohydrates and organic acids as carbon sources for growth. This study improves the knowledge on diversity, geographic distribution and evolution of bean-nodulating rhizobia in Mexico and further enlarges the spectrum of microsymbiont with which Phaseolus species can interact with, including cultivated bean varieties, notably under stressed environments. Here, the species Ensifer aridi sp. nov. is proposed as strain type of the Moroccan isolate LMR001T (= LMG 31426T; = HAMBI 3707T) recovered from desert sand dune.

Conventional and qPCR reveals the presence of 'Candidatus Liberibacter solanacearum' haplotypes A, and B in Physalis philadelphica plant, seed, and Βactericera cockerelli psyllids, with the assignment of a new haplotype H in Convolvulaceae.

The husk tomato (Physalis philadelphica Lam.) is an important Solanaceae native to Mesoamerica that is grown for its green fruit used as an important ingredient in domestic and international cuisine. Nevertheless, husk tomato plants with symptoms resembling those caused by 'Candidatus Liberibacter solanacearum' (CLso) have been observed during the last decade in plantations located in the State of Mexico, Michoacan and Sinaloa in Mexico. These areas are located near other solanaceous crops where Bactericera cockerelli the well-known psyllid transmitter of CLso is frequently present. Thus, the goal of this study was to determine if CLso haplotypes are present in husk tomato varieties in commercial fields in Mexico. From 2015 to 2016, plants and fruit showing evident symptoms of CLso infection, as well as psyllids were collected in these states and assayed by PCR for CLso using primer sets OA2/OI2c and LpFrag 1-25F/427R. Phylogenetic reconstruction was performed with Bayesian analysis and maximum likelihood methods using amplicon sequences obtained in this work along with those deposited in the GenBank database corresponding to the CLso detected in Solanaceae, Apiaceae, and Convolvulaceae host families. In addition, all the sequences were subjected to haplotype determination through an analysis of DNA polymorphisms using the DnaSP software. Furthermore, quantitative PCR (qPCR) was performed using CLso-specific primers and probes. Phylogenetic reconstruction and qPCR confirmed the presence of CLso in plants, seeds and insect-vectors, and CLso sequences from plants and seeds completely matched haplotype B, whereas CLso haplotypes A and B were detected in B. cockerelli psyllids. Polymorphism analysis identified a novel Convolvulaceae-associated CLso haplotype, which was named haplotype H. The results of this study will enable the dissemination of infected seeds to new husk tomato production areas to be avoided.

Combined application of biochar and PGPR consortia for sustainable production of wheat under semiarid conditions with a reduced dose of synthetic fertilizer.

This study investigates the combined effect of locally adopted plant growth promoting rhizobacteria (PGPR), biochar, and synthetic fertilizer on the wheat crop for the production and economic returns. A total of 20 PGPR strains were isolated from three different ecological zones of Pakistan and were evaluated. Of them, three isolates were selected for further studies. The treatments included (i) control with a full dose of the recommended fertilizer, (ii) control with half a dose of the fertilizer, (iii) PGPR consortia with half a dose of the fertilizer, (iv) biochar with half a dose of the fertilizer, and (v) PGPR + biochar with half a dose of the fertilizer. The study was repeated at three different locations. The data collected for leaf area index (LAI), grain yield, biological yield, straw yield, and harvest index (HI) revealed significant differences (P ≤ 0.05) for the locations and treatments, but the interaction of location and treatments was not significant. Based on the productivity and economic returns, the treatment with PGPR + biochar with half a dose of the fertilizer proved to be the best. Thus, the use of the PGPR consortia and biochar can improve the yield and profit of wheat crop with reduced synthetic fertilization. Graphical abstract ᅟ.

Detection of Pathogens Associated with Psyllids and Leafhoppers in Capsicum annuum L. in the Mexican States of Durango, Zacatecas, and Michoacán.

In fall 2014, 5 to 75% percent of chili and bell pepper (Capsicum annuum L.) in commercial fields located in the Mexican states of Durango, Zacatecas, and Michoacán had symptoms of deformed, small, mosaic, curled, and chlorotic leaves; shortened internodes; plant dwarfing; or phyllody and rosetting leaf tips. At the same time, leafhoppers and psyllids were observed in the fields, and more than 50 beet leafhoppers (Circulifer tenellus) and nearly 300 potato psyllids (Bactericera cockerelli) were collected from the pepper plants and adjacent weeds. Based on the insect pressure and observed symptoms, nearly 400 pepper samples were collected across this region of Mexico and tested for the presence of leafhopper- and psyllid-associated pathogens. In all, 76% of the pepper samples were found to be infected with 'Candidatus Liberibacter solanacearum', beet leafhopper-transmitted virescence agent (BLTVA) phytoplasma, a strain of a curtovirus, or a combination of any two or three of these pathogens. Additionally, 77% of the collected leafhoppers and 40% of the psyllids were infected with one or more of these pathogens, in addition to Spiroplasma citri. Specifically, the leafhoppers were infected with BLTVA phytoplasma, S. citri, or a strain of curtovirus. Of particular interest, potato psyllids were not only infected with 'Ca. L. solanacearum' but also with phytoplasmas that belong to the groups 16SrVI subgroup A and 16SrI subgroup A. The presence of mixed infections in pepper plants and the insect vectors highlights the need for growers to effectively control both leafhoppers and potato psyllids from solanaceous crops in this region of Mexico in order to prevent the spread of these bacterial and viral pathogens.

Diversity patterns of Rhizobiaceae communities inhabiting soils, root surfaces and nodules reveal a strong selection of rhizobial partners by legumes.

Current knowledge about rhizobial diversity patterns in non-nodule habitats is scarce, limiting our understanding of basic aspects of rhizobial ecology like competitiveness for nodule occupancy and host effects on community structure. We used a combination of cultivation-dependent and independent approaches to analyse alpha and beta diversity patterns of Rhizobiaceae communities from a conserved seasonally dry tropical forest site in central Mexico and two nearby agricultural fields. Lineage-specific recA amplicon libraries were generated from soil DNA and their sequences compared with those from root surface and nodule isolates recovered in trapping experiments from two native Acacia species and two Phaseolus vulgaris cultivars. Rarefaction analyses revealed that Rhizobiaceae diversity in soils is larger than on root surfaces, and smallest in nodules. A 'rare biosphere'-like distribution of species was found in the three habitats. Multivariate statistical analyses demonstrated that the plant genus exerted a stronger influence than the land-usage regime on the diversity of rhizobia associated with hosts. Rhizobium etli was the dominant Rhizobiaceae found in the soil libraries. It dominated nodulation of Acacia spp. and predominately harboured symbiovar mimosae-like nodC genes. A novel Rhizobium lineage (Rsp1) dominated bean nodulation. Specialist and generalist genotypes for host nodulation were detected in both species.

Incidence of 'Candidatus Liberibacter asiaticus'-Infected Plants Among Citrandarins as Rootstock and Scion Under Field Conditions.

Huanglongbing (HLB), caused by the bacterium 'Candidatus Liberibacter' spp., is currently one of the most serious diseases of citrus plants and has caused substantial economic losses. Thus far, there is no source of genetic resistance to HLB in the genus Citrus or its relatives. However, several studies have reported Poncirus trifoliata and some of its hybrids to be more tolerant to the disease. The main objective of this study was to report differences in the incidence of 'Ca. L. asiaticus' infection in citrandarin plants, hybrids from Sunki mandarin (Citrus sunki (Hayata) hort. ex Tanaka), and trifoliate orange Rubidoux (P. trifoliata (L.) Raf.)), after conducting an extensive survey under field conditions. These hybrid plants were established for approximately 7 years in an area with a high incidence of 'Ca. L. asiaticus'-infected plants. We selected two experimental areas (area A and area B), located approximately 10 m apart. Area A consists of Pera sweet orange (C. sinensis (L.) Osb.) grafted onto 56 different citrandarin rootstocks. Area B consists of citrandarin scions grafted onto Rangpur lime (C. limonia Osb.) rootstock. Bacteria in the leaves and roots were detected using real-time quantitative polymerase chain reaction. The incidence of 'Ca. L. asiaticus'-infected plants was 92% in area A and 14% in area B. Because infected plants occurred in both areas, we examined whether the P. trifoliata hybrid rootstock influenced HLB development and also determined the distribution of 'Ca. L. asiaticus' in Citrus tree tissues. Although this survey does not present evidence regarding the resistance of P. trifoliata and its hybrids in relation to bacteria or psyllids, future investigation, mainly using the most promising hybrids for response to 'Ca. L. asiaticus', will help us to understand the probable mechanism of defense or identifying compounds in P. trifoliata and its hybrids that are very important as strategy to combat HLB. Details of these results are presented and discussed in this article.

Liberibacter crescens gen. nov., sp. nov., the first cultured member of the genus Liberibacter.

The Gram-stain-negative, rod-shaped bacterial isolate BT-1(T) is the closest relative to the genus 'Candidatus Liberibacter' cultured to date. BT-1(T) was recovered from the phloem sap of a defoliating mountain papaya in Puerto Rico. The BT-1(T) 16S rRNA gene sequence showed that strain BT-1(T) is most closely related to members of the genus 'Ca. Liberibacter' sharing 94.7% 16S rRNA gene sequence similarity with 'Ca. Liberibacter americanus' and 'Ca. Liberibacter asiaticus'. Additionally, average nucleotide identity, 16S rRNA gene sequences and conserved protein sequences supported inclusion of the previously described species of the genus 'Ca. Liberibacter' in a genus with BT-1(T). The prominent fatty acids of isolate BT-1(T) were C18 : 1ω7c (77.2%), C16 : 0 OH (4.8%), C18 : 0 (4.4%) and C16 : 0 (3.5%). Both physiological and genomic characteristics support the creation of the genus Liberibacter, as well as the novel species Liberibacter crescens gen. nov., sp. nov. with type strain BT-1(T) ( = ATCC BAA-2481(T) = DSM 26877(T)).

Phenotypic diversity and amylolytic activity of fast growing rhizobia from pigeonpea [Cajanus cajan (L) Millsp]

This study evaluated 26 pigeonpea rhizobial isolates according to their cultural characteristics, intrinsic antibiotic resistance, salt and temperature tolerance, carbon source utilization and amylolytic activity. The cultural characterization showed that the majority of them presented the ability to acidify the YMA. Among the 27 isolates evaluated, 25 were able to grow when incubated at 42° C and 11 showed tolerance to 3% (w/v) of NaCl in YMA medium. The patterns of carbon sources utilization was very diverse among the isolates. It was observed the capacity of three strains to metabolize all the carbon sources evaluated and a total of 42% of the bacterial isolates was able to grow in the culture medium supplemented with at least, six carbon sources. The carbon sources mannitol (control) and sucrose were metabilized by all isolates evaluated. The profile of intrinsic resistance to antibiotics showed that the isolates were mostly resistant to streptomycin and ampicillin, but susceptible to kanamycin and chloranphenicol. High amylolytic activity of, at least, four isolates was also demonstrated, especially for isolated 47.3b, which showed the highest enzymatic index. These results indicate the metabolic versatility of the pigeonpea rhizobia, and indicates the isolate 47.3b to further studies regarding the amylase production and characterization.

[Quality of commercial inoculants for soybean crop in Argentina: concentration of viable rhizobia and presence of contaminants]. / Calidad de inoculantes comerciales para el cultivo de soja en la Argentina: concentración de rizobios viables y presencia de contaminantes.

In view of the inoculant production technology available, quality control is a necessary tool to improve soybean inoculants commercialized in Argentina. In 1988, the Facultad de Ciencias Agropecuarias de la Universidad Nacional de Entre Ríos (Argentina) created a quality control service for soybean crop inoculants to offer to farmers. The aim of this study was to evaluate the quality of soybean crop inoculants for seven cropping seasons and to contrast these results with those from previous investigations conducted in our country. This work was developed using 128 inoculant samples from 30 different trade names. The analyzed variables were: inoculant label information, number of viable rhizobia and presence of contaminants. Twenty per cent of the labels showed defects that did not comply with the Argentine legislation. The detected problems in inoculant labels were related to lot numbers or the expiry date, which lacked, was easy to remove or not visible. Eighty seven per cent of the analyzed inoculants were formulated in liquid carriers. Seventy six per cent of the samples had a number of rhizobia above 10(8) CFU/g or ml, the minimum quantity required by the legislation. Thirty per cent of the analyzed inoculants had contaminants and their presence was related to low rhizobia counts, as shown in a correspondence analysis. The relationship between liquid inoculants and the absence of contaminants was expressed. It can be concluded from the comparison of results found in this investigation with those in previous works published on Argentinean inoculants, that inoculant quality has been improved, although the situation is far from ideal. Adequate manufacturing and commercialization controls are necessary to ensure product quality.

Microbial community shifts influence patterns in tropical forest nitrogen fixation.

The role of biodiversity in ecosystem function receives substantial attention, yet despite the diversity and functional relevance of microorganisms, relationships between microbial community structure and ecosystem processes remain largely unknown. We used tropical rain forest fertilization plots to directly compare the relative abundance, composition and diversity of free-living nitrogen (N)-fixer communities to in situ leaf litter N fixation rates. N fixation rates varied greatly within the landscape, and 'hotspots' of high N fixation activity were observed in both control and phosphorus (P)-fertilized plots. Compared with zones of average activity, the N fixation 'hotspots' in unfertilized plots were characterized by marked differences in N-fixer community composition and had substantially higher overall diversity. P additions increased the efficiency of N-fixer communities, resulting in elevated rates of fixation per nifH gene. Furthermore, P fertilization increased N fixation rates and N-fixer abundance, eliminated a highly novel group of N-fixers, and increased N-fixer diversity. Yet the relationships between diversity and function were not simple, and coupling rate measurements to indicators of community structure revealed a biological dynamism not apparent from process measurements alone. Taken together, these data suggest that the rain forest litter layer maintains high N fixation rates and unique N-fixing organisms and that, as observed in plant community ecology, structural shifts in N-fixing communities may partially explain significant differences in system-scale N fixation rates.

rRNA operons and genome size of 'Candidatus Liberibacter americanus', a bacterium associated with citrus huanglongbing in Brazil.

Huanglongbing is one of the most severe diseases of citrus worldwide and is associated with 'Candidatus (Ca.) Liberibacter africanus' in Africa, 'Ca. Liberibacter asiaticus' in Asia and the Americas (Brazil, USA and Cuba) and 'Ca. Liberibacter americanus' (Lam) in Brazil. In the absence of axenic cultures, genetic information on liberibacters is scarce. The sequences of the entire 23S rRNA and 5S rRNA genes from Lam have now been obtained, using a consensus primer designed on known tRNAMet sequences of rhizobia. The size of the Lam genome was determined by PFGE, using Lam-infected periwinkle plants for bacterial enrichment, and was found to be close to 1.31 Mbp. In order to determine the number of ribosomal operons on the Lam genome, probes designed to detect the 16S rRNA gene and the 3' end of the 23S rRNA gene were developed and used for Southern hybridization with I-CeuI-treated genomic DNA. Our results suggest that there are three ribosomal operons in a circular genome. Lam is the first liberibacter species for which such data are available.

The tufB-secE-nusG-rplKAJL-rpoB gene cluster of the liberibacters: sequence comparisons, phylogeny and speciation.

The rplKAJL-rpoBC operon or beta operon is a classic bacterial gene cluster, which codes for proteins K, A, J and L of the large ribosomal subunit, as well as proteins B (beta subunit) and C (beta' subunit) of RNA polymerase. In the early 1990s, the operon was obtained as a 2.6 kbp DNA fragment (In-2.6) by random cloning of DNA from periwinkle plants infected with the Poona (India) strain of the huanglongbing agent, later named 'Candidatus (Ca.) Liberibacter asiaticus'. DNA from periwinkle plants infected with the Nelspruit strain (South Africa) of 'Ca. L. africanus' was amplified with a primer pair designed from In-2.6 and yielded, after cloning and sequencing, a 1.7 kbp DNA fragment (AS-1.7) of the beta operon of 'Ca. L. africanus'. The beta operon of the American liberibacter, as well as the three upstream genes (tufB, secE, nusG), have now also been obtained by the technique of chromosome walking and extend over 4673 bp, comprising the following genes: tufB, secE, nusG, rplK, rplA, rplJ, rplL and rpoB. The sequence of the beta operon was also determined for a Brazilian strain of 'Ca. L. asiaticus', from nusG to rpoB (3025 bp), and was found to share 99 % identity with the corresponding beta operon sequences of an Indian and a Japanese strain. Finally, the beta operon sequence of 'Ca. L. africanus' was extended from 1673 bp (rplA to rpoB) to 3013 bp (nusG to rpoB), making it possible to compare the beta operon sequences of the African, Asian and American liberibacters over a length of approximately 3000 bp, from nusG to rpoB. While 'Ca. L. africanus' and 'Ca. L. asiaticus' shared 81.2 % sequence identity, the percentage for 'Ca. L. americanus' and 'Ca. L. africanus' was only 72.2 %, and identity for 'Ca. L. americanus' and 'Ca. L. asiaticus' was only 71.4 %. The approximately 3000 bp nusG-rpoB sequence was also used to construct a phylogenetic tree, and this tree was found to be identical to the known 16S rRNA gene sequence-based tree. These results confirm earlier findings that 'Ca. L. americanus' is a distinct liberibacter, more distantly related to 'Ca. L. africanus' and 'Ca. L. asiaticus' than 'Ca. L. africanus' is to 'Ca. L. asiaticus'. The dates of speciation have also been estimated.

Distribution and quantification of Candidatus Liberibacter americanus, agent of huanglongbing disease of citrus in São Paulo State, Brasil, in leaves of an affected sweet orange tree as determined by PCR.

Huanglongbing (HLB), an insect-transmitted disease of citrus, known for many years in Asia and Africa, has appeared in the state of São Paulo State (SSP), Brazil, in 2004, and the state of Florida, USA, in 2005. HLB endangers the very existence of citrus, as trees infected with the bacterial pathogen, irrevocably decline. In the absence of curative procedures, control of HLB is difficult and only based on prevention. Even though not available in culture, the HLB bacterium could be shown to be Gram-negative and to represent a new candidate genus, Candidatus Liberibacter, in the alpha subdivision of the Proteobacteria. Three Candidatus (Ca.) L. species occur: Ca. L. africanus in Africa, Ca. L. asiaticus in Asia, SSP, and Florida, and Ca. L. americanus in SSP. The liberibacters occur exclusively in the phloem sieve tubes. On affected trees, HLB symptoms are often seen on certain branches only, suggesting an uneven distribution of the Liberibacter. Occurrence of Ca. L. americanus, the major HLB agent in SSP, has been examined in 822 leaf samples from an affected sweet orange tree by two conventional PCR techniques and a newly developed real time (RTi) PCR, also used for quantification of the Liberibacter in the leaves. Even though RTi-PCR was able to detect as few as 10 liberibacters per gram of leaf tissue (l/g), no liberibacters could be detected in any of the many leaf samples from a symptomless branch, while in blotchy mottle leaves from symptomatic branches of the same tree, the Liberibacter titer reached values as high as 10(7)l/g. These results demonstrate the uneven distribution of the Liberibacter in HLB-affected trees.

Molecular phylogeny based on the 16S rRNA gene of elite rhizobial strains used in Brazilian commercial inoculants.

Nitrogen is often a limiting nutrient, therefore the sustainability of food crops, forages and green manure legumes is mainly associated with their ability to establish symbiotic associations with stem and root-nodulating N2-fixing rhizobia. The selection, identification and maintenance of elite strains for each host are critical. Decades of research in Brazil resulted in a list of strains officially recommended for several legumes, but their genetic diversity is poorly known. This study aimed at gaining a better understanding of phylogenetic relationships of 68 rhizobial strains recommended for 64 legumes, based on the sequencing of the 16S rRNA genes. The strains were isolated from a wide range of legumes, including all three subfamilies and 17 tribes. Nine main phylogenetic branches were defined, seven of them related to the rhizobial species: Bradyrhizobium japonicum, B. elkanii, Rhizobium tropici, R. leguminosarum, Sinorhizobium meliloti/S. fredii, Mesorhizobium ciceri/M. loti, and Azorhizobium caulinodans. However, some strains differed by up to 35 nucleotides from the type strains, which suggests that they may represent new species. Two other clusters included bacteria showing similarity with the genera Methylobacterium and Burkholderia, and amplification with primers for nifH and/or nodC regions was achieved with these strains. Host specificity of several strains was very low, as they were capable of nodulating legumes of different tribes and subfamilies. Furthermore, host specificity was not related to 16S rRNA, therefore evolution of ribosomal and symbiotic genes may have been diverse. Finally, the great diversity observed in this study emphasizes that tropics are an important reservoir of N2-fixation genes.

'Candidatus Liberibacter americanus', associated with citrus huanglongbing (greening disease) in São Paulo State, Brazil.

Symptoms of huanglongbing (HLB) were reported in São Paulo State (SPS), Brazil, in March 2004. In Asia, HLB is caused by 'Candidatus Liberibacter asiaticus' and in Africa by 'Candidatus Liberibacter africanus'. Detection of the liberibacters is based on PCR amplification of their 16S rRNA gene with specific primers. Leaves with blotchy mottle symptoms characteristic of HLB were sampled in several farms of SPS and tested for the presence of liberibacters. 'Ca. L. asiaticus' was detected in a small number of samples but most samples gave negative PCR results. Therefore, a new HLB pathogen was suspected. Evidence for an SPS-HLB bacterium in symptomatic leaves was obtained by PCR amplification with universal primers for prokaryotic 16S rRNA gene sequences. The amplified 16S rRNA gene was cloned and sequenced. Sequence analysis and phylogeny studies showed that the 16S rRNA gene possessed the oligonucleotide signatures and the secondary loop structure characteristic of the alpha-Proteobacteria, including the liberibacters. The 16S rRNA gene sequence phylogenetic tree showed that the SPS-HLB bacterium clustered within the alpha-Proteobacteria, the liberibacters being its closest relatives. For these reasons, the SPS-HLB bacterium is considered a member of the genus 'Ca. Liberibacter'. However, while the 16S rRNA gene sequences of 'Ca. L. asiaticus' and 'Ca. L. africanus' had 98.4% similarity, the 16S rRNA gene sequence of the SPS-HLB liberibacter had only 96.0% similarity with the 16S rRNA gene sequences of 'Ca. L. asiaticus' or 'Ca. L. africanus'. This lower similarity was reflected in the phylogenetic tree, where the SPS-HLB liberibacter did not cluster within the 'Ca. L asiaticus'/'Ca. L. africanus group', but as a separate branch. Within the genus 'Candidatus Liberibacter' and for a given species, the 16S/23S intergenic region does not vary greatly. The intergenic regions of three strains of 'Ca. L. asiaticus', from India, the People's Republic of China and Japan, were found to have identical or almost identical sequences. In contrast, the intergenic regions of the SPS-HLB liberibacter, 'Ca. L. asiaticus' and 'Ca. L. africanus' had quite different sequences, with similarity between 66.0 and 79.5%. These results confirm that the SPS-HLB liberibacter is a novel species for which the name 'Candidatus Liberibacter americanus' is proposed. Like the African and the Asian liberibacters, the 'American' liberibacter is restricted to the sieve tubes of the citrus host. The liberibacter could also be detected by PCR amplification of the 16S rRNA gene in Diaphorina citri, the psyllid vector of 'Ca. L. asiaticus', suggesting that this psyllid is also a vector of 'Ca. L. americanus' in SPS. 'Ca. L. americanus' was detected in 216 of 218 symptomatic leaf samples from 47 farms in 35 municipalities, while 'Ca. L. asiaticus' was detected in only 4 of the 218 samples, indicating that 'Ca. L. americanus' is the major cause of HLB in SPS.

Development and field application of a molecular probe for the primary pathogen of the coral disease white plague type II

Abstract: One of the current problems in the field of coral disease research is that of tracking coral pathogens in the natural environment.A promising method to do this is by use of pathogen-specific molecular probes. However,this approach has been little used to date.We constructed,and validated in the laboratory,a fluoro-chrome-labeled molecular probe specific to Aurantimonas coralicida ,the bacterial pathogen of the Caribbean coral disease white plague type II (WPII).We then used the probe to test field samples of diseased coral tissue for the presence of this pathogen.Probe design was based on a unique subset (25 nucleotides)of the complete16S rRNA gene sequence derived from a pure culture of the pathogen.The pathogen-specific probe was labeled with the fluorochrome GreenStar*™FITC (fluorescein isothiocyanate,GeneDetect Ltd,New Zealand).As a control, we used the universal eubacterial probe EUB 338,labeled with a different fluorochrome (TRITC,tetra-methyl-rhodamine isothiocyanate).Both probes were applied to laboratory samples of pure cultures of bacteria, and field samples collected from the surface of the disease line of corals exhibiting signs of white plague (types I and II),healthy controls,and corals with an uncharacterized disease ("patchy necrosis ").All samples were analyzed using fluorescence in situ hybridization (FISH).We have determined that the probe is specific to our laboratory culture of the coral pathogen,and does not react with other bacterial species (the eubacterial probe does).The WPII pathogen was detected in association with diseased coral samples collected from coral colonies on reefs of the Bahamas (n=9 samples)exhibiting signs of both WPI and WPII.Diseased (and healthy)tissue samples (n=4)from corals exhibiting signs of "patchy necrosis "were also assayed.In this case the results were negative, indicating that the same pathogen is not involved in the two diseases.Incorporation and use of pathogen-specific probes can...

Citrus huanglongbing in São Paulo State, Brazil: PCR detection of the 'Candidatus' Liberibacter species associated with the disease.

Symptoms of huanglongbing (HLB), one of the most serious diseases of citrus in Asia and Africa, have been noticed in March 2004 in the Araraquara region of São Paulo State, Brazil. HLB has not been reported previously from America. The causal HLB bacteria, Candidatus Liberibacter africanus in Africa and Candidatus Liberibacter asiaticus in Asia, can be detected in symptomatic citrus leaves by PCR amplification of their 16S rDNA with previously described primers. When this technique was applied to 43 symptomatic leaf samples from the Araraquara region, all PCR reactions were negative. This suggested that a new pathogen, not detected by the above primers, could be involved in HLB in the State of São Paulo. Indeed, by using universal primers for amplification of bacterial 16S rDNA, a new liberibacter species, Candidatus Liberibacter americanus, has recently been identified. Specific primers for PCR amplification of the 16S rDNA of Ca. L. americanus have been selected. Using these primers, the new liberibacter could be detected in 214 symptomatic leaf samples tested. The leaves of two additional samples were infected with Candidatus Liberibacter asiaticus, and two further samples contained both Ca. L. americanus and Ca. L. asiaticus. The samples came from 47 farms in 35 municipalities. The psyllid vector of Ca. L. asiaticus, Diaphorina citri, is established in South, Central, and North America (Florida and Texas). Ca. L. americanus could be detected by PCR in several batches of D. citri psyllids collected on symptomatic sweet orange trees infected with Ca. L. americanus, strongly suggesting that D. citri is the vector of Ca. L. americanus. The results reported here confirm the presence of HLB in the State of São Paulo. Ca. L. americanus is the most widely distributed pathogen.

Development and field application of a molecular probe for the primary pathogen of the coral disease white plague type II.

One of the current problems in the field of coral disease research is that of tracking coral pathogens in the natural environment. A promising method to do this is by use of pathogen-specific molecular probes. However, this approach has been little used to date. We constructed, and validated in the laboratory, a fluorochrome-labeled molecular probe specific to Aurantimonas coralicida, the bacterial pathogen of the Caribbean coral disease white plague type II (WPIl). We then used the probe to test field samples of diseased coral tissue for the presence of this pathogen. Probe design was based on a unique subset (25 nucleotides) of the complete l6S rRNA gene sequence derived from a pure culture of the pathogen. The pathogen-specific probe was labeled with the fluorochrome GreenStar* FITC (fluorescein isothiocyanate, GeneDetect Ltd, New Zealand). As a control, we used the universal eubacterial probe EUB 338, labeled with a different fluorochrome (TRITC, tetra-methylrhodamine isothiocyanate). Both probes were applied to laboratory samples of pure cultures of bacteria, and field samples collected from the surface of the disease line of corals exhibiting signs of white plague (types I and II), healthy controls, and corals with an uncharacterized disease ("patchy necrosis"). All samples were analyzed using fluorescence in situ hybridization (FISH). We have determined that the probe is specific to our laboratory culture of the coral pathogen, and does not react with other bacterial species (the eubacterial probe does). The WPII pathogen was detected in association with diseased coral samples collected from coral colonies on reefs of the Bahamas (n= 9 samples) exhibiting signs of both WPI and WPII. Diseased (and healthy) tissue samples (n- 4) from corals exhibiting signs of "patchy necrosis" were also assayed. In this case the results were negative, indicating that the same pathogen is not involved in the two diseases. Incorporation and use of pathogen-specific probes can significantly expand our knowledge of the etiology of coral diseases.

Quantitative evaluation of acidity tolerance of root nodule bacteria

Quantification of acidity tolerance in the laboratory may be the first step in rhizobial strain selection for the Amazon region. The present method evaluated rhizobia in Petri dishes with YMA medium at pH 6.5 (control) and 4.5, using scores of 1.0 (sensitive, "no visible" growth) to 4.0 (tolerant, maximum growth). Growth evaluations were done at 6, 9, 12, 15 and 18 day periods. This methods permits preliminary selection of root nodule bacteria from Amazonian soils with statistical precision. Among the 31 rhizobia strains initially tested, the INPA strains 048, 078, and 671 presented scores of 4.0 at both pHs after 9 days of growth. Strain analyses using a less rigorous criterion (growth scores higher than 3.0) include in this highly tolerant group the INPA strains 511, 565, 576, 632, 649, and 658, which grew on the most diluted zone (zone 4) after 9 days. Tolerant strains still must be tested for nitrogen fixation effectiveness, competitiveness or nodules sites, and soil persistence before their recommendations as inoculants