High-resolution mapping reveals linkage between genes in common bean cultivar Ouro Negro conferring resistance to the rust,anthracnose, and angular leaf spot diseases

Key message

Co-segregation analysis and high-throughput genotyping using SNP, SSR, and KASP markers demonstrated genetic linkage between Ur-14 and Co-3 ⁴ /Phg-3 loci conferring resistance to the rust, anthracnose and angular leaf spot diseases of common bean.

Abstract

Rust, anthracnose, and angular leaf spot are major diseases of common bean in the Americas and Africa. The cultivar Ouro Negro has the Ur-14 gene that confers broad spectrum resistance to rust and the gene cluster Co-3 ⁴ /Phg-3 containing two tightly linked genes conferring resistance to anthracnose and angular leaf spot, respectively. We used co-segregation analysis and high-throughput genotyping of 179 F_2:3 families from the Rudá (susceptible) × Ouro Negro (resistant) cross-phenotyped separately with races of the rust and anthracnose pathogens. The results confirmed that Ur-14 and Co-3 ⁴ /Phg-3 cluster in Ouro Negro conferred resistance to rust and anthracnose, respectively, and that Ur-14 and the Co-3 ⁴ /Phg-3 cluster were closely linked. Genotyping the F_2:3 families, first with 5398 SNPs on the Illumina BeadChip BARCBEAN6K_3 and with 15 SSR, and eight KASP markers, specifically designed for the candidate region containing Ur-14 and Co-3 ⁴ /Phg-3, permitted the creation of a high-resolution genetic linkage map which revealed that Ur-14 was positioned at 2.2 cM from Co-3 ⁴ /Phg-3 on the short arm of chromosome Pv04 of the common bean genome. Five flanking SSR markers were tightly linked at 0.1 and 0.2 cM from Ur-14, and two flanking KASP markers were tightly linked at 0.1 and 0.3 cM from Co-3 ⁴ /Phg-3. Many other SSR, SNP, and KASP markers were also linked to these genes. These markers will be useful for the development of common bean cultivars combining the important Ur-14 and Co-3 ⁴ /Phg-3 genes conferring resistance to three of the most destructive diseases of common bean.

     

An efficient <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated transformation protocol of <Emphasis Type="Italic">Withania somnifera</Emphasis>

This is the first report on Agrobacterium rhizogenes-mediated transformation of Withania somnifera for expression of a foreign gene in hairy roots. We transformed leaf and shoot tip explants using binary vector having gusA as a reporter gene and nptII as a selectable marker gene. To improve the transformation efficiency, acetosyringone (AS) was added in three stages, Agrobacterium liquid culture, Agrobacterium infection and co-culture of explants with Agrobacterium. The addition of 75 μM AS to Agrobacterium liquid culture was found to be optimum for induction of vir genes. Moreover, the gusA gene expression in hairy roots was found to be best when the leaves and shoot tips were sonicated for 10 and 20s, respectively. Based on transformation efficiency, the Agrobacterium infection for 60 and 120 min was found to be suitable for leaves and shoot tips, respectively. Amongst the various culture media tested, MS basal medium was found to be best in hairy roots. The transformation efficiency of the improved protocol was recorded 66.5 and 59.5?% in the case of leaf and shoot tip explants, respectively. When compared with other protocols the transformation efficiency of this improved protocol was found to be 2.5 fold higher for leaves and 3.7 fold more for shoot tips. Southern blot analyses confirmed 1–2 copies of the gusA transgene in the lines W1-W4, while 1–4 transgene copies were detected in the line W5 generated by the improved protocol. Thus, we have established a robust and efficient A. rhizogenes mediated expression of transgene (s) in hairy roots of W. somnifera.     

Lima bean nodulates efficiently with <Emphasis Type="Italic">Bradyrhizobium</Emphasis> strains isolated from diverse legume species

Lima bean (Phaseolus lunatus L.) is an important legume species that establishes symbiosis with rhizobia, mainly of the Bradyrhizobium genus. The aim of this study was to evaluate the efficiency of rhizobia of the genus Bradyrhizobium in symbiosis with lima bean, in both Leonard jars and in pots with a Latossolo Amarelo distrófico (Oxisol). In the experiment in Leonard jars, 17 strains isolated from nodules of the three legume subfamilies, Papilionoideae (Vigna unguiculata, Pterocarpus sp., Macroptilium atropurpureum, Swartzia sp., and Glycine max), Mimosoideae (Inga sp.), and Caesalpinioideae (Campsiandra surinamensis) and two uninoculated controls, one with a low concentration (5.25 mg L^?1) and another with a high concentration (52.5 mg L^?1) of mineral nitrogen (N) were evaluated. The six strains that exhibited the highest efficiency in Leonard jars, isolated from nodules of Vigna unguiculata (UFLA 03–144, UFLA 03–84, and UFLA 03–150), Campsiandra surinamensis (INPA 104A), Inga sp. (INPA 54B), and Swartzia sp. (INPA 86A), were compared to two uninoculated controls, one without and another with 300 mg N dm^?3 (NH₄NO₃) applied to pots with samples of an Oxisol in the presence and absence of liming. In this experiment, liming did not affect nodulation and plant growth; the INPA 54B and INPA 86A strains stood out in terms of shoot dry matter production and provided increases of approximately 48% in shoot N accumulation compared to the native rhizobia populations. Our study is the first to indicate Bradyrhizobium strains isolated from the three legume subfamilies are able to promote lima bean growth via biological nitrogen fixation in soil conditions.     

<Emphasis Type="Italic">Mesorhizobium jarvisii</Emphasis> sv. astragali as predominant microsymbiont for <Emphasis Type="Italic">Astragalus sinicus</Emphasis> L. in acidic soils,Xinyang, China

Background and aims

Common bean (Phaseolus vulgaris L.) nodulates with a wide range of rhizobia. Amongst these is Bradyrhizobium, which is inefficient but able to induce profuse nodulation on this crop. Based on this observation, we tested whether co-inoculating bradyrhizobia with a more standard common bean symbiont, Rhizobium tropici, could stimulate growth and nodulation of common bean, thus contributing to a more effective symbiosis.

Methods

Rhizobium tropici was co-inoculated with two Bradyrhizobium strains applied at three different doses (10⁴, 10⁶, and 10⁸ CFU seed^?1) under sterile conditions, and at a single dose (10⁸ CFU seed^?1) in non-sterile soil. Plant biomass, nodulation, and N accumulation in plant tissues were evaluated.

Results

Co-inoculated plants produced more nodules, and accumulated more shoot dry biomass and nitrogen than plants inoculated with R. tropici alone under gnotobiotic conditions. Significant responses were observed at the highest inoculum dose and a significant correlation between dose and shoot dry weight was observed. Co-inoculation increased biomass and N accumulation in non-sterile soil, although with a smaller magnitude.

Conclusions

Altogether, our findings suggest that the co-inoculation with bradyrhizobia contributed to an improved symbiotic interaction between R. tropici and common beans.

     

Protein characters and relationship betweenPhaseolus vulgaris ssp.Aborigineus Burk. and related taxons of the genusPhaseolus

Both quantitative and qualitative immunochemical methods were used for studying the mutual relationships of several spocies and the subspecies of the genusPhaseolus: Ph. vulgaris L. ssp.vulgaris, Ph. vulgaris L. ssp.aborigineus Burk.,Ph. coccineus L.,Ph. acutifolius A. Gray,Ph. lunatus L. (American endemites) andPh. aureus L. (a typical Asian bean). Protein characters of cotyledons (i.e., ?storage” proteins) of the above species were compared with the aid of antisera prepared against seed (cotyledon) proteins ofPh. vulgaris L. ssp.vulgaris, cv. Veltruská Saxa, using

1. (a)
   the whole complex of cotyledon protein,     
   
   

Karyotypic study of five Lutjanid species using conventional and Ag-NORs banding techniques

The first cytogenetic comparisons of five snapper species from Thailand were presented here. Renal cell samples were taken from blacktail snapper (Lutjanus fulvus), five lined snapper (L. quinquelineatus), dory snapper (L. fulviflamma), brownstripe red snapper (L. vitta), and mangrove red snapper (L. argentimaculatus). The mitotic chromosome preparation was prepared directly from kidney cells. Conventional staining and Ag-NOR banding techniques were applied to stain the chromosomes. The results exhibited that all five snapper species have the diploid chromosome numbers of 2n = 48 and the fundamental numbers (NF) of 48. The presences of large, medium, and small telocentric chromosomes were 22-24-2, 24-20-4, 36-10-2, 28-16-4 and 36-10-2, respectively. The Ag- NORs banding technique provides the pair of nucleolar organizer regions (NORs) at subcentromeric region of the long arm of the respective telocentric chromosome pairs 9, 1, 3, 4 and 9. Their karyotype formulas is as follows: L. fulvus (2n = 48): L ₂₂ ^t + M ₂₄ ^t + S ₂ ^t , L. quinquelineatus (2n = 48): L ₂₄ ^t + M ₂₀ ^t + S ₄ ^t , L. fulviflamma (2n = 48): Lt36 + Mt10 + St2, L. vitta (2n = 48): L ₂₈ ^t + M ₁₆ ^t + S ₄ ^t , and L. argentimaculatus (2n = 48): L ₃₆ ^t + M ₁₀ ^t + S ₂ ^t .     

Detection and Quantification of <Emphasis Type="Italic">Vibrio cholerae,Vibrio parahaemolyticus</Emphasis>, <Emphasis Type="Italic">and Vibrio vulnificus</Emphasis> in Coastal Waters of Guinea-Bissau (West Africa)

V. cholerae, V. parahaemolyticus, and V. vulnificus are recognized human pathogens. Although several studies are available worldwide, both on environmental and clinical contexts, little is known about the ecology of these vibrios in African coastal waters. In this study, their co-occurrence and relationships to key environmental constraints in the coastal waters of Guinea-Bissau were examined using the most probable number-polymerase chain reaction (MPN-PCR) approach. All Vibrio species were universally detected showing higher concentrations by the end of the wet season. The abundance of V. cholerae (ISR 16S-23S rRNA) ranged 0–1.2 × 10⁴ MPN/L, whereas V. parahaemolyticus (toxR) varied from 47.9 to 1.2 × 10⁵ MPN/L. Although the presence of genotypes associated with virulence was found in environmental V. cholerae isolates, ctxA+ V. cholerae was detected, by MPN-PCR, only on two occasions. Enteropathogenic (tdh+ and trh+) V. parahaemolyticus were detected at concentrations up to 1.2 × 10³ MPN/L. V. vulnificus (vvhA) was detected simultaneously in all surveyed sites only at the end of the wet season, with maximum concentrations of 1.2 × 10⁵ MPN/L. Our results suggest that sea surface water temperature and salinity were the major environmental controls to all Vibrio species. This study represents the first detection and quantification of co-occurring Vibrio species in West African coastal waters, highlighting the potential health risk associated with the persistence of human pathogenic Vibrio species.     

Purification and characteristics of a novel bacteriocin produced by <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> L11 isolated from Chinese traditional fermented cucumber

Objective

To purify and characterize a novel bacteriocin with broad inhibitory spectrum produced by an isolate of Enterococcus faecalis from Chinese fermented cucumber.

Results

E. faecalis L11 produced a bacteriocin with antimicrobial activity against both Escherichia coli and Staphylococcus aureus. The amino acid sequence of the purified bacteriocin, enterocin L11, was assayed by Edman degradation method. It differs from other class II bacteriocins and exhibited a broad antimicrobial activity against not only Gram-positive bacteria, including Bacillus subtilis, S. aureus, Listeria monocytogenes, Sarcina flava, Lactobacillus acidophilus, L. plantarum, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. bulgaricus and Streptococcus thermophilus, but also some Gram-negative bacteria including Salmonella typhimurium, E. coli and Shigella flexneri. Enterocin L11 retained 91 % of its activity after holding at 121 °C for 30 min. It was also resistant to acids and alkalis.

Conclusions

Enterocin L11 is a novel broad-spectrum Class II bacteriocin produced by E. faecalis L11, and may have potential as a food biopreservative.

     

Molecular authentication of two medicinal plants <Emphasis Type="Italic">Ligularia fischeri</Emphasis> and <Emphasis Type="Italic">Ligularia stenocephala</Emphasis> using allele-specific PCR (AS-PCR) strategy

Ligularia fischeri (Gom-chi) and Ligularia stenocephala (Gon-dal-bi) are popular edible herbs in Korea. L. fischeri is used to treat jaundice, hepatitis, rheumatoid arthritis, and scarlet fever, while L. stenocephala is used to treat anxiety, weakness, and menstrual disorders. The herbal medicinal activities of these two herbs differ, but they are very difficult to distinguish based on their morphologies, especially in their dried forms. In an effort to distinguish these two plant species, we sequenced three barcoding genes in plastids, matK, rbcL, and trnH-psbA. From the analysis of sequence variations, we detected five single nucleotide polymorphisms (SNPs) between two the species. Allele specific (AS)-primers in the SNPs were employed in discrimination of the two species. Of the five AS-primer sets, one primer pair in matK gene showed reproducibly distinguishable PCR amplification between plants of L. fischeri and L. stenocephala. The method is reproducible and efficient, and is the first reported molecular method to discriminate between L. fischeri and L. stenocephala.     

The C-terminus of DSX<Superscript>F5</Superscript> protein acts as a novel regulatory domain in <Emphasis Type="Italic">Bombyx mori</Emphasis>

The doublesex gene regulates the somatic sexual development of Bombyx mori by alternatively splicing into sex-specific splice forms. In our previous study, the splice form Bmdsx ^F7 , which encodes the BmDSX^F5 protein, was found to be expressed in a female-specific manner and to contain a novel C-terminus. In this study, we aimed to investigate the role of this C-terminus. Two transgenic lines, L1 and L2, were constructed to ectopically express Bmdsx ^F7 in males. Phenotype and W chromosome-specific polymerase chain reaction (PCR) analysis showed that developmental abnormalities and sex reversal did not occur. Moreover, the sex ratio was also normal. Quantitative PCR revealed that the expression levels of SP1 and Vg were upregulated in the fat body of transgenic males. Additionally, the expression level of PBP was downregulated in the antenna of transgenic males. The results suggested that the C-terminus of BmDSX^F5 functioned as a regulatory domain during regulation of downstream target gene expression and that BmDSX^F5 participated in the sexual development of somatic cells together with other DSX proteins in B. mori.     

<Emphasis Type="Italic">Rhizobium</Emphasis> strains in the biological control of the phytopathogenic fungi <Emphasis Type="Italic">Sclerotium</Emphasis> (<Emphasis Type="Italic">Athelia</Emphasis>) <Emphasis Type="Italic">rolfsii</Emphasis> on the common bean

Aims

To identify Rhizobium strains’ ability to biocontrol Sclerotium rolfsii, a fungus that causes serious damage to the common bean and other important crops, 78 previously isolated rhizobia from common bean were assessed.

Methods

Dual cultures, volatiles, indole-acetic acid (IAA), siderophore production and 16S rRNA sequencing were employed to select strains for pot and field experiments.

Results

Thirty-three antagonistic strains were detected in dual cultures, 16 of which were able to inhibit ≥84% fungus mycelial growth. Antagonistic strains produced up to 36.5 μg mL^?1 of IAA, and a direct correlation was verified between IAA production and mycelium inhibition. SEMIA 460 inhibited 45% of mycelial growth through volatile compounds. 16S rRNA sequences confirmed strains as Rhizobium species. In pot condition, common bean plants grown on S. rolfsii-infested soil and inoculated with SEMIA 4032, 4077, 4088, 4080, 4085, or 439 presented less or no disease symptoms. The most efficient strains under field conditions, SEMIA 439 and 4088, decreased disease incidence by 18.3 and 14.5% of the S. rolfsii-infested control.

Conclusions

Rhizobium strains could be strong antagonists towards S. rolfsii growth. SEMIA 4032, 4077, 4088, 4080, 4085, and 439 are effective in the biological control of the collar rot of the common bean.

     

Biochemical evidence bearing on the domestication of<Emphasis Type="Italic">Phaseolus</Emphasis> (Fabaceae) beans

The genusPhaseolus (Fabaceae) consists of some 50 species, all of which are distributed in the Americas. Four of these contain cultigens.P. vulgaris (common bean),P. lunatus (lima bean),P. acutifolius (tepary bean),P. coccineus subsp.coccineus (runner bean); andP. coccineus subsp.polyanthus (no English vernacular name). Biochemical markers—phaseolin seed storage protein and isozymes—have provided new evidence on the organization of the first three species. Domestication has possibly caused a strong reduction in genetic diversity inP. vulgaris andP. acutifolius. BothP. vulgaris andP. lunatus cultivars result from at least two independent domestications, in Mesoamerica and in the Andes. These two species consist of two gene pools, each of which includes wild ancestors and their respective cultivated descendants. Our findings suggest the need for additional emphasis on genetic conservation of wild ancestors and their use in breeding programs and for a comparison of inter-gene pool vs. intra-gene pool crosses in breeding programs.     

<Emphasis Type="Italic">Lecanicillium muscarium</Emphasis> and <Emphasis Type="Italic">Adalia bipunctata</Emphasis> combination for the control of black bean aphid<Emphasis Type="Italic">, Aphis fabae</Emphasis>

The predator Adalia bipunctata (Coleoptera: Coccinellidae) and the entomopathogenic fungus Lecanicillium muscarium, have been considered as potential biological control against aphids. While it can be difficult to achieve a high control level of Aphis fabae Scopoli (Hemiptera: Aphididae) using only a single beneficial agent, the research presented here aimed to determine the interaction between L. muscarium and A. bipunctata potential for control against A. fabae. Lecanicillium muscarium was found to cause about 30% mortality in A. bipunctata and with a reduction in feeding by about 15%. However, co-application of A. bipunctata and L. muscarium can cause an addititive effect in reducing aphid populations, resulting in about 90% reduction in aphid populations compared with control treatment. Thus, these two biocontrol agents have the potential to be complementary. This research study demonstrates that it is possible to combine A. bipunctata with L. muscarium to provide a sustainable method for management of A. fabae on broad bean cropping system and that field studies are required.     

Laboratory and simulated-field bioassays for assessing mixed cultures of <Emphasis Type="Italic">Lysinibacillus sphaericus</Emphasis> against <Emphasis Type="Italic">Aedes aegypti</Emphasis> (Diptera: Culicidae) larvae resistant to temephos

Aedes aegypti (L.) is the main vector of tropical diseases such as dengue, chikungunya and Zika. Due to the overuse of insecticides, Ae. aegypti resistant populations have increased. Biological control with Lysinibacillus sphaericus (Ahmed) has been used against Culex sp. and Anopheles sp. Although Ae. aegypti is refractory to the binary toxin of L. sphaericus spores, vegetative cells have been shown to be effective against Ae. aegypti larvae. In this work, the effect of L. sphaericus vegetative cells on Ae. aegypti temephos-resistant larvae was assessed under lab and simulated field conditions. L. sphaericus caused about 90% mortality of insecticide-resistant Ae. aegypti larvae under simulated field conditions. Likewise, Ae. aegypti larvae were more sensitive to mixed cultures of L. sphaericus than to individual strains; then, the most effective mixed culture exhibited an LC₅₀ of 1.21 × 10⁵ CFU/mL with Rockefeller larvae and 8.04 × 10⁴ CFU/mL with field-collected larvae. Additionally, we found that mixed cultures composed of two L. sphaericus strains were more effective than a culture formed by the three strains. Our results suggest that mixed cultures comprising L. sphaericus vegetative cells could be useful for controlling temephos-resistant populations of Ae. aegypti, as evidenced by the effectiveness demonstrated under laboratory and simulated field conditions.     

Development and validation of a robust,breeder-friendly molecular marker for the <Emphasis Type="Italic">vc</Emphasis><Superscript>-</Superscript> locus in faba bean

Faba bean (Vicia faba L.) is a valuable feed and food crop with potential for development globally as a staple protein crop. Its consumption is limited by the anti-nutritional factors vicine and convicine (v-c) in its seeds. A single gene (vc ^- ) confers the low v-c phenotype in faba bean. Time-consuming and laborious quantitative chemical analysis is currently used in breeding programs to detect v-c concentration. Molecular markers within or linked to the vc ^- gene could facilitate rapid and cost-effective screening of early generation breeding populations for low v-c concentration. The large and complex faba bean genome has been an impediment to the progress of development of molecular breeding strategies. Here, we report a high-throughput low-cost KASP (Kompetitive Allele Specific PCR) marker for low v-c concentration in faba bean. The KASP assay successfully distinguished low and high v-c lines of faba bean. This marker is a significant and valuable molecular tool for faba bean breeding programs aiming to reduce v-c from faba beans worldwide.     

Mung bean (<Emphasis Type="Italic">Vigna radiata</Emphasis>) cultivars mediated oviposition preference and development of <Emphasis Type="Italic">Callosobruchus chinensis</Emphasis> (Coleoptera: Chrysomelidae: Bruchinae)

The Azuki bean weevil, Callosobruchus chinensis (L.), is a destructive pest of stored mung bean [Vigna radiata (L.) Wilczek] as well as other leguminous seeds. The development of resistant seeds to manage this pest is of current great interest to plant breeders. In this study, we investigated the oviposition preference and development of C. chinensis on two susceptible mung bean cultivars (Seonhwa and Gyeongseon) and one previously reported resistant cultivar (Jangan), compared to the susceptible cowpea (Vigna unguiculata L.), cultivar (Yeonbun) using both multiple-choice and no-choice tests. In addition, the development of C. chinensis was also examined at four constant temperatures (20, 25, 30, and 35 °C). Both tests found cowpea to be the most suitable seed for oviposition. Total developmental time from oviposition to adult emergence ranged from 27.01 to 38.2 days, being shortest on cowpea and longest on the mung bean, cv. Jangan. However, no successful development of C. chinensis larvae on mung bean, cv. Jangan, occurred at any temperature. The highest rate of adult emergence and the longest adult longevity both occurred on cowpea and certain mung bean cultivars (Seonhwa and Gyeongseon), with the dramatic exception of cv. Jangan. These results suggest that the higher preference and performance of C. chinensis on cowpea (3.3 egg/seed) and least on mung bean, cv. Jangan (0.4 egg/seed). This information may facilitate the exploration of resistant genetic materials and chemicals associated with seeds for successful breeding. Further studies should examine the chemicals associated with mung bean cultivars and its resistant mechanism to develop a control method against bruchines.     

A physiological comparative study of acid tolerance of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> ZDY 2013 and <Emphasis Type="Italic">L. plantarum</Emphasis> ATCC 8014 at membrane and cytoplasm levels

This study aimed to disclose the acid tolerance mechanism of Lactobacillus plantarum by comparing L. plantarum ZDY 2013 with the type strain L. plantarum ATCC 8014 in terms of cell membrane, energy metabolism, and amino acid metabolism. L. plantarum ZDY 2013 had a superior growth performance under acidic condition with 100-fold higher survival rate than that of L. plantarum ATCC 8014 at pH 2.5. To determine the acid tolerance physiological mechanism, cell integrity was investigated through scanning electron microscopy. The study revealed that L. plantarum ZDY 2013 maintained cell morphology and integrity, which is much better than L. plantarum ATCC 8014 under acid stress. Analysis of energy metabolism showed that, at pH 5.0, L. plantarum ZDY 2013 enhanced the activity of Na⁺/K⁺-ATPase and decreased the ratio of NAD⁺/NADH in comparison with L. plantarum ATCC 8014. Similarly, amino acid metabolism of intracellular arginine, glutamate, and alanine was improved in L. plantarum ZDY 2013. Correspondingly, the activity of arginine deiminase and glutamate decarboxylase of L. plantarum ZDY 2013 increased by 1.2-fold and 1.3-fold compared with L. plantarum ATCC 8014 in acid stress. In summary, it is demonstrated that the special physiological behaviors (integrity of cell membrane, enhanced energy metabolism, increased amino acid and enzyme level) of L. plantarum ZDY 2013 can protect the cells from acid stress.     

Detection of <Emphasis Type="Italic">Legionella</Emphasis> by quantitative-polymerase chain reaction (qPCR) for monitoring and risk assessment

Background

Culture and quantitative polymerase chain reaction (qPCR) assays for the detection of Legionella were compared on samples from a residential area before and after two interventions. A total of 84 samples were collected from shower hoses and taps as first flush samples and at constant temperature. Samples were grouped according to the origin of the sample, a) circulation water b) water from empty apartments c) water from shower hoses. The aims were to investigate the usefulness of qPCR compared to culture for monitoring remedial actions for elimination of Legionella bacteria and as a tool for risk assessment.

Results

In water collected from the apartments Legionella spp were detected by qPCR in the concentration range from LOQ to 9.6*10⁵GU/L while L. pneumophila were detected in a range from LOQ to 6.8*10⁵ GU/L. By culturing, the legionellae were detected in the range from below detection limit (> 10 CFU/L) to 1.6*10⁶ CFU/L. In circulating water and in first flush water from shower hoses, culture and qPCR showed the same tendencies. The overall correlation between the bacteria number detected by culture and the two developed qPCR assays (L. spp and L. pneumophila) was relatively poor (r² = 0.31 for culture and Legionella spp. assay, r² = 0.20 for culture and L. pneumophila assay).

Conclusion

Detection by qPCR was suitable for monitoring changes in the concentration of Legionella but the precise determination of bacteria is difficult. Risk assessment by qPCR only on samples without any background information regarding treatment, timing, etc is dubious. However, the rapid detection by qPCR of high concentrations of Legionella - especially Legionella pneumophila - is valuable as an indicator of risk, although it may be false positive compared to culture results. On the other hand, the detection of a low number of bacteria by qPCR is a strong indication for the absence of risk.