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ObjectiveErwinia amylovora causes fire blight disease in Malus. A strong resistance QTL (Mfu10) was previously detected on linkage group 10 of Malus fusca accession MAL0045, using several strains of the bacterium. As no strain capable of breaking the resistance of MAL0045 has been found, it was hypothesized that a second resistance factor contributes to the fire blight resistance of MAL0045. However, to date, no minor locus has been detected with previously published strains of the bacterium. We detected a minor QTL only on a subset of a population following inoculation with strain Ea1038, which heterologously expresses an effector in a derivative of isolate Ea3049. Two genetic maps of MAL0045, one scarce, the other dense with markers, were used for QTL analyses.ResultsMfu10 was detected on LG10 with Ea1038, as was previously with Ea3049. Although no other QTLs of significant LOD was previously detected in other linkage groups with Ea3049, a QTL of significant LOD was detected on LG16 (Mfu16) after inoculation of a subset of 76 individuals with Ea1038, but only using the dense genetic map. Mfu16 improved the effect of Mfu10. However, when the number of individuals inoculated with Ea1038 was increased to 121, Mfu16 was no longer detected in the dense genetic map. We hypothesize some factors, which might be responsible for the instability of this QTL.

In continuing previous work on the role of AAA+ proteins of the apple proliferation agent ‘Candidatus Phytoplasma mali’ in phytoplasma virulence and suppression of virulence, 147 full-length deduced protein sequences of AAA+ ATPase AP460 of single- and multiple-strain accessions were examined. This approach revealed that in this protein two regions can be distinguished. Region 1, located in the N-terminal part, is characterized by 14 highly conserved substitutions associated with the suppression of virulence. However, these substitutions were not present in all attenuated strains. In the more diverse region 2, located in the C-terminal part, highly conserved substitutions associated with two groups of virulence were identified. In addition to the virulence-related residues, three other groups of conserved substitutions are present in strains with attenuated virulence with or without the presence of suppression-associated substitutions in region 1. In one of these groups, substitutions next to key residues of the ATPase motifs sensor 1 and 2 and arginine finger do occur that seem to affect ATPase function. This group of substitution was present in all attenuated strains. From these findings it can be concluded that two different mechanisms of suppression exist of which the supposed effect on ATPase function seems to be more important than the suppression-associated substitutions in region 1. The presence of virulent, avirulent and suppressive strains in a tree leads in the commonly occurring multiple infections to interactions in which the resulting virulence is determined by the numerical relation of virulent and attenuated strains.

Bacteria were isolated from necrotic apple and pear tree tissue and from dead wood in Germany and Austria as well as from pear tree exudate in China. They were selected for growth at 37 °C, screened for levan production and then characterized as Gram-negative, facultatively anaerobic rods. Nucleotide sequences from 16S rRNA genes, the housekeeping genes dnaJ, gyrB, recA and rpoB alignments, BLAST searches and phenotypic data confirmed by MALDI-TOF analysis showed that these bacteria belong to the genus Gibbsiella and resembled strains isolated from diseased oaks in Britain and Spain. Gibbsiella-specific PCR primers were designed from the proline isomerase and the levansucrase genes. Acid secretion was investigated by screening for halo formation on calcium carbonate agar and the compound identified by NMR as acetic acid. Its production by Gibbsiella spp. strains was also determined in culture supernatants by GC/MS analysis after derivatization with pentafluorobenzyl bromide. Some strains were differentiated by the PFGE patterns of SpeI digests and by sequence analyses of the lsc and the ppiD genes, and the Chinese Gibbsiella strain was most divergent. The newly investigated bacteria as well as Gibbsiella querinecans, Gibbsiella dentisursi and Gibbsiella papilionis, isolated in Britain, Spain, Korea and Japan, are taxonomically related Enterobacteriaceae, tolerate and secrete acetic acid. We therefore propose to unify them in the species Gibbsiella acetica sp. nov.

Fire blight, caused by enterobacterium Erwinia amylovora, is the most important bacterial disease affecting pome fruit. It can cause devastating economic losses and is reliably controlled only by the application of antibiotics, which are banned in many European countries due to environmental and sustainability issues and consumers’ mistrust. One solution could be the utilization of fire blight resistant cultivars in apple production. In 2003, we started an approach at Dresden-Pillnitz to detect different mechanisms conferring resistance to fire blight aimed at their com-bination in new cultivars. Four segregating populations were established to map QTLs for fire blight resistance. The donors used were three wild species accessions of Malus baccata, M. fusca, M. ×robusta 5 and the Pillnitz cultivar ‘Rewena’. The susceptible parent in each case was Idared. Grafted scions of each progeny were inoculated with E. amylovora strain Ea222_JKI. Average percent lesion length (PLL) of all progenies was determined. Genetic linkage maps were established using DArT- SCAR-, SNP-, and SSR-markers. Whereas in Rewena no QTL could be determined, major QTLs were detected in M. baccata on linkage group 12, in M. fusca on linkage group 10, and in M. ×robusta 5 on linkage group 3 explaining up to around 50, 85 and 85% of the phenotypic variance, respectively. The fact that all resistance QTLs are located on different linkage groups enhances the chance that different mechanisms are acting in the donors. Additionally, trees of the Idared by M. ×robusta 5 population were planted in an orchard and flowers were inoculated for phenotyping. The QTL on linkage group 3 could be con-firmed after mapping.

Flower infections caused by Erwinia amylovora can be prevented only during the short period of epiphytic colonization. Bacterial antagonists like Erwinia tasmaniensis and Bacillus spp. are able to repress E. amylovora growth at this initial stage and prevent further intrusion of the pathogen. They can be applied as biological control agents if a suitable formulation can be produced. To investigate inhibitory effects of different antagonists and dried formulations, we used several test systems in the laboratory, including co-cultivation experiments, agar diffusion assay and detached-flower assays. A luminescent reporter strain of E. amylovora was used to distinguish between pathogen and antagonist growth. In laboratory studies, application of dry formulations was as efficient as with antagonists derived from batch culture. Samples from test-formulations were also applied in field trials according to EPPO guideline 1/166.

The angular leaf spot disease caused by Xanthomonas fragariae is an important plant disease with major impact for the strawberry nursery industry. Currently there is no plant protection product available for controlling the disease effectively. Planting of resistant cultivars seems to be promising, but all commercially used cultivars are susceptible and no donor with a high level of resistance has yet been found. Therefore, a total of 145 genotypes from the Fruit Genebank Dresden (Germany) were evaluated for resistance to X. fragariae by artificial inoculation. Six genotypes were classified as partly resistant, out of which only two (US4808 and US4809) are octoploid. Fragaria vesca f. alba, Fragaria nilgerrensis ‘Yunnan’, F. vesca ‘Illa Martin’ and F. moschata ‘Bauwens’ were also classified as partially resistant, but they are only of limited use for breeding because of their variable ploidy level. Fully resistant genotypes could not be detected. The systemic dispersal of the bacteria in strawberry plants was investigated after inoculation of leaves with X. fragariae strain XF3.9.C and the GFP-tagged strain XF3.9.C(pKAN). The systemic spread was evaluated after 3, 7, 14 and 28 days post-inoculation (dpi) by nested PCR and fluorescence microscopy. After 3 dpi, X. fragariae could be found in all tissues tested including the inoculated leaf, its petiole, the rhizome, the heart bud up to the youngest fully expanded leaf and its petiole. The systemic spread was also detectable in partially resistant genotypes.

We identified a compound in culture supernatants of Erwinia species, such as Erwinia amylovora, E. pyrifoliae, E. billingiae, E. tasmaniensis, E. persicina and E. rhapontici absorbing at 340 nm, which was associated before with the yellow pigment produced by E. amylovora on media containing copper ions. The compound was purified from E. tasmaniensis strain Et1/99 supernatants by chromatography on Dowex-1 and Dowex-50 columns and identified by HPLC/MS and NMR analysis as 6-thioguanine (6TG). Its signal at 167 Da matched with the expected molecular mass. By random mutagenesis with miniTn5, we obtained mutants defective in the genes for pyrimidine and purine metabolism. A specific gene cluster with ycf genes described by us before, absent in the corresponding region of Escherichia coli, was identified in the genome sequence of three Erwinia species and named tgs region for thioguanine synthesis. Clones of the tgs gene cluster promoted 6TG synthesis and secretion in E. coli, when the bacteria were grown in minimal medium supplemented with amino acids. 6TG was bacteriostatic for E. coli and Salmonella typhimurium strains, with cell growth resumed after prolonged incubation. Similar results were obtained with P. agglomerans strains. Bacteria from the genus Pectobacterium were barely and Rahnella or Gibbsiella species were not inhibited by 6TG. Adenine and guanine relieved the toxic effect of 6TG on E. coli. Non-producing strains were fully virulent on host plants. 6TG synthesis may help erwinias to interfere with growth of some microorganisms in the environment.

Summary Fire blight is a destructive bacterial disease caused by Erwinia amylovora affecting plants in the family Rosaceae, including apple. Host resistance to fire blight is present mainly in accessions of Malus spp. and is thought to be quantitative in this pathosystem. In this study we analyzed the importance of the E. amylovora effector avrRpt2EA, a homolog of Pseudomonas syringae avrRpt2, for resistance of Malus × robusta 5 (Mr5). The deletion mutant E. amylovora Ea1189ΔavrRpt2EA was able to overcome the fire blight resistance of Mr5. One single nucleotide polymorphism (SNP), resulting in an exchange of cysteine to serine in the encoded protein, was detected in avrRpt2EA of several Erwinia strains differing in virulence to Mr5. E. amylovora strains encoding serine (S-allele) were able to overcome resistance of Mr5, whereas strains encoding cysteine (C-allele) were not. Allele specificity was also observed in a coexpression assay with Arabidopsis thaliana RIN4 in Nicotiana benthamiana. A homolog of RIN4 has been detected and isolated in Mr5. These results suggest a system similar to the interaction of RPS2 from A. thaliana and AvrRpt2 from P. syringae with RIN4 as guard. Our data are suggestive of a gene-for-gene relationship for the host–pathogen system Mr5 and E. amylovora.

The epiphyte Pantoea agglomerans 48b/90 (Pa48b) is a promising biocontrol strain against economically important bacterial pathogens such as Erwinia amylovora. Strain Pa48b produces the broad-spectrum antibiotic 2-amino-3-(oxirane-2,3-dicarboxamido)-propanoyl-valine (APV) in a temperature-dependent manner. An APV-negative mutant still suppressed the E. amylovora population and fire blight disease symptoms in apple blossom experiments under greenhouse conditions, but was inferior to the Pa48b wild-type indicating the influence of APV in the antagonism. In plant experiments with the soybean pathogen Pseudomonas syringae pv. glycinea both, Pa48b and the APV-negative mutant, successfully suppressed the pathogen. Our results demonstrate that the P. agglomerans strain Pa48b is an efficient biocontrol organism against plant pathogens, and we prove its ability for fast colonization of plant surfaces over a wide temperature range.

Aims: To detect the apple and pear pathogens Erwinia amylovora and Erwinia pyrifoliae as well as the related epiphytes Erwinia tasmaniensis and Erwinia billingiae, we created novel PCR primers and also applied them to a series of other plant-associated bacteria as control. To facilitate fast diagnosis, we used matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI–TOF MS). Methods and Results: The PCR primers were deduced from the pstS–glmS regions, which can include the gene for levansucrase, and also from regions encoding capsular polysaccharide synthesis. All primer combinations were specific for their associated Erwinia species to detect them with conventional PCR, also in mixed cultures from necrotic plant tissue. Other primers designed for quantitative PCR with SYBR Green or together with TaqMan probes were applied for real-time detection to determine growth of Erw. amylovora, Erw. billingiae, Erw. pyrifoliae and Erw. tasmaniensis in apple blossoms. From whole-cell protein extracts, profiles were generated using a Bruker microflex machine and Erwinia strains classified according to a score scheme. Conclusions: The designed PCR primers identified the Erwinia species unambiguously and can be applied to qualitative and quantitative tests. MALDI–TOF MS data were in agreement with the PCR assays. Significance and Impact of the Study: The applied diagnosis methods allow fast and precise monitoring of two pathogenic and two epiphytic Erwinia species. They are valuable for population studies with apple and pear flowers and with diseased plant material.

Erwinia amylovora produces a compound with an absorbance maximum at 340 nm that forms a yellow colored complex with copper. This compound was identified as 6-thioguanine, a guanine analogue that is used in chemotherapy and the treatment of inflammatory bowel disease. Synthesis of 6-thioguanine could be linked to five genes common in Erwinia genomes, but missing in related genera. Expression of Erwinia tasmaniensis genes tgsA-D in Escherichia coli was sufficient for heterologous production of 6-thioguanine. Transfer of the four biosynthetic E. tasmaniensis genes did not enhance resistance of E. coli against 6-thioguanine. Bacterial and synthetic 6-thioguanine have a strong growth inhibitory effect on many bacteria, such as E. coli or a number of Pantoea agglomerans isolates. While this inhibition of competing species might provide an advantage to Erwinia, further biological functions of 6-thioguanine cannot be excluded. A direct link between 6-thioguanine synthesis of E. amylovora and its pathogenicity was not observed, as two 6-thioguanine negative mutants produced as severe symptoms on apple and pear shoots as did producing strains.

The harlequin ladybird Harmonia axyridis (Coleoptera: Coccinellidae) is a polyphagous predatory beetle native to Central and Eastern Asia. Since 2007 it has established all over Central Europe. In order to elucidate which defense strategy is responsible for its high resistance to diseases, we tested hemolymph as well as eleven main components of the headspace of H. axyridis for antimicrobial activity against Gram-positive (Bacillus subtilis, B. thuringiensis ssp. tenebrionis, Micrococcus luteus) and Gram-negative bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae). While three of the volatile compounds weakly reduced the growth of microorganisms, hemolymph of adults and larvae of H. axyridis strongly inhibited the growth of Gram-positive and Gram-negative bacteria as well as yeast. Furthermore, we compared the antimicrobial activity in the hemolymph of H. axyridis and Coccinella septempunctata. Antimicrobial activity in H. axyridis was about a thousand times higher compared to hemolymph from C. septempunctata. In contrast to C. septempunctata, the antimicrobial activity in H. axyridis was present without prior challenge. Minimal inhibitory concentration (MIC) of the hemolymph of H. axyridis was lowest against E. coli and yeast followed by B. subtilis, and was highest against entomopathogenic B. thuringiensis ssp. tenebrionidae. Furthermore, MIC values of the hemolymph obtained from live beetles were significantly lower than from frozen insects. This suggests that the active antimicrobial compound is affected by freezing and subsequent thawing of the beetles. Potential implications of our findings for the competitive advantages of H. axyridis over C. septempunctata are discussed.

The epiphyte Pseudomonas syringae pv. syringae 22d / 93 (Pss22d), isolated from soybean leaves, had been characterized as a promising and species-specific biocontrol strain in vitro and in planta against the plant pathogen P. syringae pv. glycinea (Psg), which causes bacterial blight of soybean. Three toxins are known to be produced by Pss22d: syringomycin, syringopeptin and 3-methylarginine (MeArg). In contrast to syringopeptin and syringomycin, MeArg inhibited the growth of Psg in vitro. To examine if the toxins produced by Pss22d are responsible for antagonistic effects in planta, the pathogen Psg was co-inoculated with either Pss22d wild-type, a syringopeptin/syringomycin-negative double mutant (Pss22d.ΔsypA/syrE), or a MeArg-negative mutant (Pss22d.1) into wounds of pin-pricked leaves of greenhouse-grown soybean plants, respectively. In all three cases, the wild-type Pss22d and its toxin-deficient mutants prevented development of disease symptoms normally caused by Psg. These results indicated that neither syringopeptin, nor syringomycin, nor MeArg was required for Pss22d's antagonistic activity in planta. Consequently, factors other than the three toxins may contribute to the intra-species antagonism in planta.

For fast and cost-efficient identification of bacteria we tested the Biotyper system (Bruker Daltonics) to detect Erwinia amylovora and other plant-associated bacteria by protein profiles from whole cells. Strains could also be identified by direct transfer of cells to the sample target, and the signals were compared to preceding protein extraction with formic acid. The identification scores obtained for extracted proteins were higher and more reliable than those of directly applied agar cultured cells. For verification of MALDI-TOF identification and for detection of several Erwinia species in mixed cultures we applied PCR primer sets designed from specific chromosomal sequence regions such as the gene cluster for biosynthesis of capsular EPS or the nucleotide sequences flanked by genes pstS and glmS. The novel primers readily detected E. amylovora fruit tree and raspberry strains, E. pyrifoliae, E. billingiae and E. tasmaniensis. The identification obtained by PCR agreed with the MALDI-TOF MS results. While PCR analysis is also applicable for detection of bacteria in mixed cultures obtained from plant samples, MALDI-TOF analysis identifies essentially cells from single colonies applied from agar plates or cultured in liquid medium. Both methods were combined for identification of bacteria in field samples and from artificially inoculated plant material.

Short-chain fatty acids (SCFAs) are used to preserve food and could be a tool for control of fire blight caused by Erwinia amylovora on apple, pear and related rosaceous plants. Neutralized acids were added to buffered growth media at 0.5–75 mM and tested at pHs ranging from 6.8 to 5.5. Particularly at low pH, SCFAs with a chain length exceeding that of acetic acid such as propionic acid were effective growth inhibitors of E. amylovora possibly due to uptake of free acid and its intracellular accumulation. We also observed high inhibition with monochloroacetic acid. An E. billingiae strain was as sensitive to the acids as E. amylovora or E. tasmaniensis. Fire blight symptoms on pear slices were reduced when the slices were pretreated with neutralized propionic acid. Propionic acid is well water soluble and could be applied in orchards as a control agent for fire blight.

Glycine betaine is an effective osmoprotectant for Bacillus subtilis. Its import into osmotically stressed cells led to the buildup of large pools, whose size was sensitively determined by the degree of the osmotic stress imposed. The amassing of glycine betaine caused repression of the formation of an osmostress-adaptive pool of proline, the only osmoprotectant that B. subtilis can synthesize de novo. The ABC transporter OpuA is the main glycine betaine uptake system of B. subtilis. Expression of opuA was upregulated in response to both sudden and sustained increases in the external osmolarity. Nonionic osmolytes exerted a stronger inducing effect on transcription than ionic osmolytes, and this was reflected in the development of corresponding OpuA-mediated glycine betaine pools. Primer extension analysis and site-directed mutagenesis pinpointed the osmotically controlled opuA promoter. Deviations from the consensus sequence of SigA-type promoters serve to keep the transcriptional activity of the opuA promoter low in the absence of osmotic stress. opuA expression was downregulated in a finely tuned manner in response to increases in the intracellular glycine betaine pool, regardless of whether this osmoprotectant was imported or was newly synthesized from choline. Such an effect was also exerted by carnitine, an effective osmoprotectant for B. subtilis that is not a substrate for the OpuA transporter. opuA expression was upregulated in a B. subtilis mutant that was unable to synthesize proline in response to osmotic stress. Collectively, our data suggest that the intracellular solute pool is a key determinant for the osmotic control of opuA expression.

Fire blight is a destructive bacterial disease caused by Erwinia amylovora affecting plants in the family Rosaceae, including apple. Host resistance to fire blight is present mainly in accessions of Malus spp. and is thought to be quantitative in this pathosystem. In this study we analyzed the importance of the E. amylovora effector avrRpt2(EA) , a homolog of Pseudomonas syringae avrRpt2, for resistance of Malus × robusta 5 (Mr5). The deletion mutant E. amylovora Ea1189ΔavrRpt2(EA) was able to overcome the fire blight resistance of Mr5. One single nucleotide polymorphism (SNP), resulting in an exchange of cysteine to serine in the encoded protein, was detected in avrRpt2(EA) of several Erwinia strains differing in virulence to Mr5. E. amylovora strains encoding serine (S-allele) were able to overcome resistance of Mr5, whereas strains encoding cysteine (C-allele) were not. Allele specificity was also observed in a coexpression assay with Arabidopsis thaliana RIN4 in Nicotiana benthamiana. A homolog of RIN4 has been detected and isolated in Mr5. These results suggest a system similar to the interaction of RPS2 from A. thaliana and AvrRpt2 from P. syringae with RIN4 as guard. Our data are suggestive of a gene-for-gene relationship for the host-pathogen system Mr5 and E. amylovora.

Pantoea stewartii subsp. stewartii is the causative agent of Stewart's wilt, a bacterial disease transmitted by the corn flea beetle mainly to sweet corn ( Zea mays ). In many countries, it is classified as a quarantine organism and must be differentiated from other yellow enteric bacteria frequently occurring with corn. We have created novel primers from the pstS-glmS region of P. stewartii for use in conventional PCR (cPCR) and quantitative PCR (qPCR). To facilitate rapid diagnosis, we applied matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Using whole-cell protein extracts, profiles were generated with a Bruker microflex machine, and the bacteria classified. P. stewartii strains were clearly distinguished from strains of Pantoea agglomerans , Pantoea dispersa , and Pantoea ananatis . Dendrogram analysis of the protein profiles confirmed the score values and showed the formation of separate clades for each species. The identification achieved by MALDI-TOF MS analysis agrees with the diagnosis by specific PCR primers. The combination of both methods allows a rapid and simple identification of the corn pathogen. P. stewartii subsp. stewartii and P. stewartii subsp. indologenes are highly related and can be distinguished not only by virulence assays and indole tests but also by a characteristic pattern in the nucleotide sequence of recA .

The use of naturally occurring microbial antagonists to suppress plant diseases offers a favorable alternative to classical methods of plant protection. The soybean epiphyte Pseudomonas syringae pv. syringae strain 22d/93 shows great potential for controlling P. syringae pv. glycinea, the causal agent of bacterial blight of soybean. Its activity against P. syringae pv. glycinea is highly reproducible even in field trials, and the suppression mechanisms involved are of special interest. In this work we demonstrated that P . syringae pv. syringae 22d/93 produced a significantly larger amount of siderophores than the pathogen P. syringae pv. glycinea produced. While P . syringae pv. syringae 22d/93 and P. syringae pv. glycinea produce the same siderophores, achromobactin and pyoverdin, the regulation of siderophore biosynthesis in the former organism is very different from that in the latter organism. The epiphytic fitness of P . syringae pv. syringae 22d/93 mutants defective in siderophore biosynthesis was determined following spray inoculation of soybean leaves. The population size of the siderophore-negative mutant P . syringae pv. syringae strain 22d/93ΔSid was 2 orders of magnitude lower than that of the wild type 10 days after inoculation. The growth deficiency was compensated for when wound inoculation was used, indicating the availability of iron in the presence of small lesions on the leaves. Our results suggest that siderophore production has an indirect effect on the biocontrol activity of P . syringae pv. syringae 22d/93. Although siderophore-defective mutants of P . syringae pv. syringae 22d/93 still suppressed development of bacterial blight caused by P. syringae pv. glycinea, siderophore production enhanced the epiphytic fitness and thus the competitiveness of the antagonist.

The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces the rare amino acid 3-methylarginine (MeArg), which is highly active against the closely related soybean pathogen Pseudomonas syringae pv. glycinea. Since these pathogens compete for the same habitat, Pss22d is a promising candidate for biocontrol of P. syringae pv. glycinea. The MeArg biosynthesis gene cluster codes for the S -adenosylmethionine (SAM)-dependent methyltransferase MrsA, the putative aminotransferase MrsB, and the amino acid exporter MrsC. Transfer of the whole gene cluster into Escherichia coli resulted in heterologous production of MeArg. The methyltransferase MrsA was overexpressed in E. coli as a His-tagged protein and functionally characterized ( K m , 7 mM; k cat , 85 min −1 ). The highly selective methyltransferase MrsA transfers the methyl group from SAM into 5-guanidino-2-oxo-pentanoic acid to yield 5-guanidino-3-methyl-2-oxo-pentanoic acid, which then only needs to be transaminated to result in the antibiotic MeArg.

A strong fire blight resistance QTL (Mfu10) was previously detected on linkage group 10 (LG10) of Malus fusca accession MAL0045, using several strains of the causative bacterium, Erwinia amylovora. As no strain capable of breaking the resistance of MAL0045 has been found, we hypothesized that another locus contributes to its fire blight resistance. However, none was detected with strains previously tested on the progeny. Here, an avrRpt2EA mutant strain (Ea1038) with the chromosomal S-allele deleted and complemented with the less aggressive C-allele, was used to phenotype MAL0045 × ‘Idared’ progeny. We performed phenotype-genotype analyses using the first genetic map of MAL0045, which is scarcely dense, and a recently constructed saturated map. As expected, Mfu10 was detected on LG10 with Ea1038, as was previously with other strains. Interestingly, a QTL with a logarithm of odds (LOD) thresholds of 5.5 and 2.9, significant at the genome-wide and chromosome levels, respectively, was detected with Ea1038 on LG16 (Mfu16) in a subset of 76 individuals, but only using the saturated map. Progenies carrying both Mfu10 and Mfu16 were significantly more resistant than progenies carrying only Mfu10. However, the LOD of Mfu16 diminished to 2.6 in a larger subset of individuals. We hypothesize that Mfu16 is present in the genome of MAL0045 albeit unstable in the progeny.