Hyalophora cecropia pupae were infected by Enterobacter cloacae C7‐501 to induce antibacterial attacins for purification. The induction of attacins in immunized pupae was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). Ion‐exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), and Rotofor® isoelectric focusing (ISEF) were applied to isolate attacins from the hemolymph. IEC separated attacins from most hemolymph proteins, but the fractions containing attacins also had other proteins of 20 and 64 kDa in length. In IEC, attacin was eluted with ~0.2 M NaCl. The best conditions for IEC were pH 9, flow rate of 2 mL/min, with step elution (0.025, 0.05, 0.075, 0.1, 0.2, 0.4 and 1.0 M NaCl). In HIC, most other proteins were eluted with the ammonium persulfate treatment. HIC isolated attacin proteins under hydrophobic conditions, at ~50% EtOH. However, the fraction with attacins also contained other proteins. The Rotofor® ISEF produced fractions containing attacins at isoelectric points ranging between 5.7 and 8.3. However, non‐specific proteins were detected in the fraction samples, and the recovery of attacins was low. The purification efficiency of ISEF was lower than IEC and HIC. In this study, the expression of attacins was induced in H. cecropia pupae infected with E. cloacae C7‐501, and attacins could be purified by IEC and ISEF. Overall, IEC provided better separation of attacins from the hemolymph of H. cecropia pupae immunized with E. cloacae bacteria than HIC and Rotofor® ISEF.

The use of biocontrol agents and natural compounds as alternatives to synthetic fungicides was a relatively obscure topic of research until the mid-1980s, but this field has flourished in recent years. This chapter provides a comprehensive overview of pre- and postharvest biological approaches to manage postharvest diseases of fruit crops, including the use of biological agents and microbial fermentation products, physical treatments, plant extracts, and genetic markers for host resistance that can be utilized in breeding programs.

Summary Plants evolved in association with a diverse community of microorganisms. The effect of plant phylogeny and domestication on host–microbiome co‐evolutionary dynamics are poorly understood. Here we examined the effect of domestication and plant lineage on the composition of the endophytic microbiome of 11 Malus species, representing three major groups: domesticated apple (M. domestica), wild apple progenitors, and wild Malus species. The endophytic community of M. domestica and its wild progenitors showed higher microbial diversity and abundance than wild Malus species. Heirloom and modern cultivars harbored a distinct community composition, though the difference was not significant. A community‐wide Bayesian model revealed that the endophytic microbiome of domesticated apple is an admixture of its wild progenitors, with clear evidence for microbiome introgression, especially for the bacterial community. We observed a significant correlation between the evolutionary distance of Malus species and their microbiome. This study supports co‐evolution between Malus species and their microbiome during domestication. This finding has major implications for future breeding programs and our understanding of the evolution of plants and their microbiomes.

Breeding apple cultivars with resistance offers a potential solution to fire blight, a damaging bacterial disease caused by Erwinia amylovora . Most resistance alleles at quantitative trait loci (QTLs) were previously characterized in diverse Malus germplasm with poor fruit quality, which reduces breeding utility. This study utilized a pedigree-based QTL analysis approach to elucidate the genetic basis of resistance/susceptibility to fire blight from multiple genetic sources in germplasm relevant to U.S. apple breeding programs. Twenty-seven important breeding parents (IBPs) were represented by 314 offspring from 32 full-sib families, with ‘Honeycrisp’ being the most highly represented IBP. Analyzing resistance/susceptibility data from a two-year replicated field inoculation study and previously curated genome-wide single nucleotide polymorphism data, QTLs were consistently mapped on chromosomes (Chrs.) 6, 7, and 15. These QTLs together explained ~28% of phenotypic variation. The Chr. 6 and Chr. 15 QTLs colocalized with previously reported QTLs, while the Chr. 7 QTL is possibly novel. ‘Honeycrisp’ inherited a rare reduced-susceptibility allele at the Chr. 6 QTL from its grandparent ‘Frostbite’. The highly resistant IBP ‘Enterprise’ had at least one putative reduced-susceptibility allele at all three QTLs. In general, lower susceptibility was observed for individuals with higher numbers of reduced-susceptibility alleles across QTLs. This study highlighted QTL mapping and allele characterization of resistance/susceptibility to fire blight in complex pedigree-connected apple breeding germplasm. Knowledge gained will enable more informed parental selection and development of trait-predictive DNA tests for pyramiding favorable alleles and selection of superior apple cultivars with resistance to fire blight.

Fire blight, caused by the bacterial pathogen Erwinia amylovora, is a persistent problem for pear (Pyrus spp.) growers in the United States. Growing resistant cultivars is one of the best options for managing fire blight. The cultivars Potomac and Old Home and the selection NJA2R59T69 display resistance to fire blight. As such, three mapping populations (El Dorado × Potomac, Old Home × Bartlett, and NJA2R59T69 × Bartlett) were developed to identify genomic regions associated with resistance to fire blight. Progeny were phenotyped during 2017 and 2018 by inoculating multiple actively growing shoots of field-grown seedling trees with E. amylovora isolate E153n via the cut-leaf method. Genotyping was conducted using the recently developed Axiom Pear 70 K Genotyping Array and chromosomal linkage groups were created for each population. An integrated two-way pseudo-testcross approach was used to map quantitative trait loci (QTLs). Resistance QTLs were identified on chromosome 2 for each population. The QTLs identified in the El Dorado × Potomac and Old Home × Bartlett populations are in the same region as QTLs that were previously identified in Harrow Sweet and Moonglow. The QTL in NJA2R59T69 mapped proximally to the previously identified QTLs and originated from an unknown Asian or occidental source. Future research will focus on further characterizing the resistance regions and developing tools for DNA-informed breeding.

Apple industries suffer from major apple diseases because of widely planted susceptible cultivars. Developed disease-resistant cultivars that often carry only a single source of resistance are not expected to be durable over time. Cultivars with multiple sources of resistance are often commercially unacceptable due to unsatisfactory fruit quality alleles inherited from unimproved and improved parents. To improve fruit quality, approximately five modified backcrossing generations have been used with phenotypic selection for offspring with the least proportion of unimproved genome and elite fruit quality. Modified backcrossing is time-consuming owing to the long juvenility of apple. Unimproved parents are always assumed to carry undesirable alleles in addition to the targeted resistance allele. To efficiently identify favorable offspring each generation, DNA-based markers would be useful. Locus-specific DNA tests are unavailable to detect many sources of resistance alleles. Known numbers of DNA segments from unimproved parents could help subsequent parent selection among offspring because of the direct connection to the probability of eliminating such segments each generation. Accurately estimating the proportion and number of unimproved segments requires precise information on genomic positions of recombinations that can be detected with effective genetic marker sets. High-resolution and genome-wide apple SNP arrays can be used to characterize unimproved DNA segments present in disease-resistant offspring to efficiently achieve durable resistance and elite fruit quality. To hasten apple flowering, a rapid generation cycling approach with transgenic genetic stocks is available. Using these tools is expected to effectively exploit additional unimproved germplasm toward apple genetic improvement.

Malus sieversii is considered the progenitor of modern apple (Malus pumila) cultivars and to represent a valuable source of genetic diversity. Despite the importance of M. sieversii as a source of disease resistance, stress tolerance, and novel fruit traits, little is known about gene function and diversity in M. sieversii. Notably, a publicly annotated genome sequence for this species is not available. In the current study, the FOX (Full-length cDNA OvereXpressing) gene hunting system was used to construct a library of transgenic lines of Arabidopsis in which each transgenic line overexpresses a full-length gene obtained from a cDNA library of the PI619283 accession of M. sieversii. The cDNA library was constructed from mRNA obtained from bark tissues collected in late fall–early winter, a time at which many abiotic stress-adaptative genes are expressed. Over 4000 apple FOX Arabidopsis lines have been established from the pool of transgenic seeds and cDNA inserts corresponding to various Gene Ontology (GO) categories have been identified. A total of 160 inserts appear to be novel, with no or limited homology to M. pumila, Arabidopsis, or poplar. Over 1300 lines have also been screened for freezing resistance. The constructed library of transgenic lines provides a valuable genetic resource for exploring gene function and diversity in Malus sieversii. Notably, no such library of t-DNA lines currently exists for any Malus species.

Apple blue mold causes significant postharvest economic losses worldwide. A blue mold resistance locus, qM-Pe3.1, was previously identified on chromosome 3 of Malus sieversii PI 613981, a wild accession with inferior fruit quality. Introgression of the resistance allele into elite breeding germplasm is difficult and success of introgression and the effect of the PI 613981 genome on fruit quality cannot be phenotypically evaluated until fruiting, which occurs approximately 5 years from seed. In this study, introgression of the qM-Pe3.1 resistance allele was achieved by rapid cycle breeding, utilizing the transgenic line T1190 constitutively expressing the BpMADS4 early-flowering gene. This was supported by DNA-based diagnostic information that enabled marker-assisted selection for blue mold resistance using a locus-specific DNA test developed to detect the qM-Pe3.1 resistance allele in offspring (foreground selection). Of 75 second-generation ([‘Gala’ × PI 613981] × T1190) offspring carrying BpMADS4, 43 also carried the qM-Pe3.1 resistance allele. DNA tests for other trait loci were used to identify other desirable alleles related to fruit quality in progeny and 6874 genome-wide SNP markers (from an apple 20K Illumina® SNP array) were used to identify undesirable genomic segments of PI 613981 (background selection). Three individuals identified with favorable recombination close to qM-Pe3.1 and less than 25% of M. sieversii unimproved genome were selected as best suited for the elimination of unimproved DNA segments in subsequent generations. Our pipeline for introgression of qM-Pe3.1, facilitated by marker-assisted foreground and background selection, successfully advanced this promising germplasm in readiness for the next generation.

The article was published with errors in Table 1 and Online Resource 1 found in the results section and supplementary material.

Fire blight ( Erwinia amylovora ), a potentially devastating disease in apple, can cause floral, fruit and structural damage and even tree death. Most commercial apple cultivars are susceptible and the resistance/susceptibility of many modern cultivars has not been evaluated. Fire blight resistance/susceptibility is difficult to phenotype due to quantitative resistance, impacts of tree vigour and environment on susceptibility, and the erratic nature of the disease. Resistance/susceptibility levels were determined for 94 apple cultivars and important breeding parents. In 2016 and 2017, multiple actively growing shoots per tree (about three trees per cultivar) were challenged with E. amylovora Ea153n via a cut‐leaf inoculation method. Proportion of current season's shoot length blighted ( SLB ) was calculated for each shoot. To classify cultivar responses, estimated marginal SLB means were compared to four controls, representing highly susceptible ( HS ) to highly resistant ( HR ), via Dunnett's tests. Cultivar responses ranged from HS to HR with estimated marginal SLB means of 0.001–0.995 in 2016 and 0.000–0.885 in 2017. Most cultivars demonstrated similar resistance/susceptibility levels in both years (ρ = 0.657, P < 0.0001). K‐means clustering was used to classify cultivars into three resistance/susceptibility groups based on incidence, average severity ( SLB ), and maximum severity values (maximum SLB and age of wood infected). Sixteen cultivars were consistently moderately resistant ( MR ) to HR while the remainder ranged from HS to MR . An updated comparison of susceptibility of important cultivars is provided. Resistance/susceptibility information gained could be used to identify genetic loci associated with resistance/susceptibility and/or inform parental selection in apple scion breeding programmes.

Fire blight disease, caused by the bacterium Erwinia amylovora, has developed to an economical important disease in cultivation of pome fruits in many regions of the world. The bacterial cysteine protease AvrRpt2EA was identified as a central molecule in the host-pathogen interaction and it is important for pathogen recognition in the fire blight resistant crabapple Malus ×robusta 5. However, little is known about its role as virulence factor in susceptible apples. To investigate its function in planta, transgenic lines of the fire blight-susceptible cultivar ‘Pinova’ were generated, which contain an plant-optimized version of AvrRpt2EA driven by a heat shock-inducible promoter. After induced expression of AvrRpt2EA, the transgenic lines showed symptoms similar to natural fire blight infections, such as shoot necrosis and browning of older leaves. Furthermore, an increase of the expression of the PR-1 gene was shown, which was used as molecular marker for salicylic acid (SA) dependent systemic acquired resistance (SAR). Additional analysis reveal that the levels of SA and its derivatives were increased after AvrRpt2EA expression, too, with diverse kinetics in leaves of different ages. In contrast, no induction of the expression level of VSP2 paralogs was found, which were used as marker genes for the activation of the jasmonic acid (JA)-dependent defense pathway. This was also confirmed by metabolic profiling of JA and its derivatives. In conclusion, the results of this study show that AvrRpt2EA alone acts as virulence factor causing fire blight disease symptoms in susceptible apple plants and induces the formation of SA and SA-dependent SAR.

Apple trees have a long juvenile period, which makes apple genetic improvement via breeding costly and time-consuming. Transgenic genetic stocks carrying the early-flowering gene BpMADS4 have been used to reduce the juvenility of apple from five or more years to less than 10months. One such genetic stock, T1190, has been used widely in breeding and research. It was reported to be a seedling of Pinova' but the other parent was unknown. Not knowing the alleles that this unknown parent contributed to T1190 brings uncertainties to breeding programs and research studies. In this study, the full pedigree of the genetic stock T1190 was reconstructed using an apple 20K SNP array and a panel of 530 reference cultivars and breeding selections. T1190 was determined to be an offspring of Pinova' and Idared'. The full pedigree of T1190 was used to deduce the mosaic ancestor composition of the transgene-hosting chromosome. Such knowledge is useful to ensure breeding programs and research studies achieve their expected objectives.

Die Feuerbrandkrankheit, ausgelost durch das Bakterium Erwinia amylovora , hat sich in den letzten Jahrzehnten in vielen Regionen der Erde zu einer okonomisch bedeutenden Krankheit im Kernobstanbau entwickelt. In der Wirt-Pathogen-Beziehung spielt das Effektorprotein AvrRpt2 EA des bakteriellen Erregers eine zentrale Rolle. So ist AvrRpt2 EA in der Feuerbrand-resistenten Wildart des Apfels Malus x robusta 5 fur die Erkennung des Pathogens wichtig, hingegen ist uber die Wirkungsweise des Effektorproteins in anfalligen Apfelgenotypen noch wenig bekannt. Um die Funktion von AvrRpt2 EA in planta zu untersuchen, wurde eine fur die pflanzliche Proteinbiosynthese optimierte Version des bakteriellen AvrRpt2 EA Gens mit Hilfe der Agrobakterien -vermittelten Transformation in das Genom der anfalligen Sorte 'Pinova' eingebracht. Zur Regulation der AvrRpt2 EA Genexpression wurde der Hitzeschock-induzierbare Gmhsp17.5-E Promoter von Glycine max verwendet. Nach der induzierten Expression von AvrRpt2 EA in den transgenen Linien bildeten sich Nekrosen im Bereich des Triebs und Verbraunungen alterer Blatter aus, die mit der typischen Symptomatik einer naturlichen Feuerbrandinfektion vergleichbar sind. Die transgene Expression des Effektorproteins fuhrte in den Linien zu einer Induktion der Genexpression des Salicylsaure (SA)-regulierten Gens PR-1 , das die Aktivierung der Systemisch erworbene Resistenz (engl. systemic acquired resistance, SAR) anzeigt. Durch metabolische Analysen konnte ebenfalls ein Anstieg der Level an SA und SA-Derivate mit unterschiedlichen Kinetiken abhangig vom Blattalter nachgewiesen werden. Hingegen konnte kein Anstieg des Expressionslevels von VSP2 Paralogen detektiert werden, welche als Markergene fur die Aktivierung des Jasmonsaure (JA)-abhangigen Abwehrwegs genutzt wurden. Diese Beobachtung konnte weiterhin durch die metabolische Messung von JA und deren Derivate bestatigt werden. Zusammenfassend demonstrieren die Untersuchungen, dass AvrRpt2 EA in einem anfalligen Apfelgenotyp als Virulenzfaktor agiert und dort die Bildung von SA und den SA-abhangigen Weg der SAR induziert.

Penicillium expansum is a major postharvest pathogen that infects different fruits, mainly through injuries inflicted during harvest or subsequent handling after harvest. Several effectors were suggested to mediate pathogenicity of P. expansum in fruit tissue. Among these effectors Nep1-like proteins (NLPs), produced by various microorganisms with different lifestyles, are known for their ability to induce necrosis in dicot plants and were shown to be involved in virulence of several plant-related pathogens. This study was aimed at the identification and functional characterization of two NLP genes found in the genome of P. expansum. The genes were designated Penlp1 and Penlp2 and were found to code type1 and type3 NLP respectively. Necrosis-inducing activity of the two proteins was demonstrated by transient expression in Nicotiana benthamiana leaves. While Penlp1 expression was induced during apple infection and in liquid culture, the highest level of Penlp2 expression was found in ungerminated spores. Deletion of Penlp1, but not Penlp2, resulted in reduced virulence on apples manifested by reduced rate of lesion development (disease severity).