Powdery mildews (PM) are common and severe pathogen groups that threaten plants, and PM resistance is complex and polygenic in cucumbers. Previously mlo-based resistance was reported in various plants, including cucumber, with generated loss-of CsaMLO function mutants. However, mlo-based resistance in cucumber is also complex and involves additional mechanisms such as hypersensitive response (HR) and papillae formation. For this reason, we focused on determining the mlo-based powdery mildew resistance mechanism in cucumber. CRISPR/Cas9 was used in the present study to generate loss-of-function mutants for CsaMLO1, CsaMLO8, and CsaMLO11 of PM susceptible ADR27 cucumber inbred lines and CsaMLO mutants were obtained and validated. Trypan Blue and DAB staining were performed to detect Podosphaera xanthii germination/penetration rates and accumulation of Reactive Oxygen Species (ROS). Our results indicate that PM-susceptibility associated CsaMLOs in cucumber are negative regulators in different defense mechanisms against powdery mildew at early and late stages of infection. Further, the experiment results indicated that CsaMLO8 mutation-based resistance was associated with the pre-invasive response, while CsaMLO1 and CsaMLO11 could be negative regulators in the post-invasive defense response in cucumber against P. xanthii. Although the loss-of CsaMLO8 function confers the highest penetration resistance, CsaMLO1 and CsaMLO11 double mutations could be potential candidates for HR-based resistance against PM pathogen in cucumber. These results highlighted the crucial role of CRISPR/Cas9 to develop PM resistant cucumber cultivars, possessing strong pre-invasive defense with CsaMLO8 or post-invasive with CsaMLO1/CsaMLO11 mutations.

CRISPR/Cas9 is one of the most robust technologies for plant breeding enabling precise and efficient modifications in a genome. This technology is being used for the manipulation of target genes in a host to develop resistance against the plant pathogens. Cucumis sativus elF4E is one of the target genes playing a key role in viral infection during interaction with potyvirus viral proteins genome linked (VPg). Nevertheless, the allelic and positional effect of elF4E mutations in C. sativus is to be clarified in elF4E-VPg interaction. In addition, there are entanglements in the massive production of pathogen-resistant cultivars suitable for commercial production using CRISPR/Cas9 technology. Therefore, we targeted different positions of the elF4E in G27 and G247 inbred lines, using specific gRNA1 and gRNA2 for the first and third exons, respectively, and 1,221 transgene-free plants were selected in segregated T1 generation, where 192 G27 and 79 G247 plants had the least mutation at Cas9 cleavage site of gRNA1 or gRNA2. Crossing was performed to see allelic effects of elfF4E mutations in F1 populations, which were homozygous and heterozygous single (elF4E_1^DEL or elF4E_3^DEL) and double (elF4E_1-3^DEL) mutants. Disease symptoms of watermelon mosaic virus (WMV), papaya ringspot virus (PRSV), and zucchini yellow mosaic virus (ZYMV) were evaluated in both non-edited and edited F1 plants, and we did not observe any symptom in homozygous elF4E_1-3^DEL and elF4E_1^DEL mutants. However, homozygous elF4E_3^DEL was positive in reverse transcription polymerase chain reaction (RT-PCR), even if there were no significant symptoms on the inoculated leaves. ELISA and qRT-PCR indicated lower viral accumulation in homozygous elF4E_3^DEL than heterozygous and non-edited plants. Regeneration and transformation protocols were also optimized comprehensively for both the genotypes. The average number of shoots/100 explants was determined for both G27 and G247 as 13.6 and 18.0, respectively. We could not detect any distinguishing difference between the non-edited and edited F1 plants for yield and morphology. Our results demonstrate an effective route for mass production of viral resistant cultivars of cucumber to WMV, ZYMV, and PRSV. In this way, the pathogen-resistant cultivars could be generated to reduce the losses caused by these pathogens in cucumber production.

Podosphaera xanthii causes powdery mildew of cucumber, and is associated with significant yield and quality losses. Development of resistant or tolerant varieties is the most effective and eco-friendly strategy for powdery mildew management. An important host resistance mechanism is based on the recognition of conserved resistance genes, resulting in durable resistance. To determine powdery mildew resistance mechanisms in cucumber, total RNAs were isolated from the powdery mildew resistant cultivar Meltem, the tolerant line VT18, and the susceptible local variety Camlica. Expression levels of nine genes in these plants were analysed by Reverse Transcription Polymerase Chain Reaction (RT-PCR). The host reactions were assessed using microscope observations of stained specimens. Serine/threonine (STN7), transcription factor (WRKY22), serine/threonine-protein kinase (D6PKL1), and serine/threonine receptor kinase (NFP) genes were induced, as positive regulators in defence mechanisms against powdery mildew. Polygalacturonase Inhibitor (PGIP) did not express after P. xanthii inoculation of Camlica, resulting in susceptibility. After inoculation, callose synthase (CALLOSE) and cinnamyl alcohol dehydrogenase (CAD) gene expression levels were increased in resistant Meltem, but Hypersensitive Reaction (HR) and ROS formation were only linked in the tolerant VT18. Powdery mildew development was less in Meltem than in VT18, indicating that cell wall thickening and HR play separate roles in resistance to this disease.

Avocado (Persea americana Milll.) holds a pivotal position in global fruit crops, contributing significantly to the economies of tropical and subtropical regions. However, the rising incidence of diseases poses a substantial risk to avocado production. This comprehensive study investigated the disease landscape in Antalya, the largest avocado cultivation area in the Türkiye. A survey of 2537 avocado trees across 11 regions from 2020 to 2021 revealed alarming disease incidences, particularly in the eastern regions of Gazipasa and Alanya. Dieback, branch canker, anthracnose, and soil-borne root rot were identified as the primary diseases affecting tree canopies, twigs, and branches. Morphological and molecular analyzes unveiled a spectrum of pathogens, with Colletotrichum gloeosporioides dominating in the Mediterranean region. Notably, Phytophthora cinnamomi emerged as a severe threat, causing root rot and decline in avocado trees. Fusarium solani and Fusarium oxysporum, known for their association with tropical fruit crops, were identified in the western parts of Antalya. Additionally, we have detected Neofusicoccum parvum, Lasiodiplodia theobromae, and Neopestalotiopsis rosae in collected samples from avocado trees. The identified pathogens exhibited varying levels of severity in branch canker and anthracnose on avocado branches and leaves. Furthermore, pathogenicity evaluations shed light on the potential of these pathogens to induce severe symptoms, emphasizing the urgency for effective control measures. The exploration of cultural and biological control strategies are crucial for mitigating the impact of branch canker, dieback, and anthracnose diseases, ensuring the sustainability of avocado cultivation in the region.

Erwinia amylovora causes fire blight mainly on pear, apple and quince trees. This bacterial pathogen also infects other Rosaceous plants, such as blackberry and raspberry. A race structure was established between an isolate of E. amylovora and berries using 40 wild blackberry and 7 wild raspberry genotypes. In pathogenicity tests, wild blackberry and raspberry genotypes had three phenotypic reactions: enhanced susceptibility, moderate susceptibility and resistance. We noted a higher bacterial growth of over 300 × 10⁹ cfu mL^-1 in plants with enhanced susceptibility, with resistant genotypes showing a bacterial growth of around 150 × 10⁹ cfu mL^-1. These results are also associated with symptoms observed at 29 days post-inoculation. This resistance is being evaluated to control fire blight.