First Report of Crown Rot Caused by Fusarium algeriense on Wheat in Kyrgyzstan.

Fusarium crown rot of wheat is an economically important disease that leads to significant yield and quality losses, especially in many arid and semi-arid wheat-growing areas worldwide. In June 2020, winter wheat (Triticum aestivum L.) plants exhibiting crown rot symptoms were identified in a commercial field located in the Tokbay location (43.033719°N, 74.325623°E), Chuy Province, Kyrgyzstan. The diseased plants were stunted and had brown discoloration on internodes of the stem bases and roots. Disease incidence was about 3%. A total of 10 plants were sampled at the ripening stage from the field to identify the causal agent. Symptomatic tissues were excised, surface disinfected with 1% NaOCl, rinsed three times with distilled water, and placed on one-fifth strength potato dextrose agar (PDA) followed by incubation at 23°C in the dark for 5 days. A total of 8 Fusarium isolates were recovered from tissues and purified by the hyphal tips method onto fresh PDA and Spezieller-Nährstoffarmer agar (SNA) plates (Leslie and Summerell 2006). Sequence analysis of the translation elongation factor 1α (TEF1) and the RNA polymerase II beta subunit (RPB2) genes were performed with primers EF1 and EF2 (O'Donnell et al. 1998), and 5f2 (Reeb et al. 2004) and 7cr (Liu et al. 1999), respectively. The sequences of three isolates showed 100% identities with the corresponding sequences of the strain NRRL 66652 of Fusarium algeriense Laraba & O'Donnell (TEF1: MF120515 and RPB2: MF120504), and the sequences of a representative isolate (KyrFa01) were deposited in GenBank (TEF1: OM135603 and RPB2: OM135604). On PDA, fungal colonies were initially yellowish-white but gradually turned yellowish-brown. Ellipsoidal microconidia produced in false heads on monophialides were usually aseptate (8.30 ± 1.17 µm, n = 50) and occasionally one-septate (21.89 ± 2.01 µm, n = 50). Sporodochial macroconidia were mostly 3-4 septate measuring 43.41 ± 2.83 µm (n = 50), slightly curved and formed generally on monophialides on SNA. No chlamydospores formation was detected after 15 days on SNA or PDA. Morphological characteristics described above were consistent with the morphology of F. algeriense, as reported by Laraba et al. (2017). To confirm pathogenicity, seeds of wheat cultivar Seri 82, Fusarium crown rot susceptible, were treated in 1% NaOCl for 2 min, rinsed twice, and placed in plates containing a piece of sterile filter paper saturated with water to induce germination for 3 days. Five pregerminated seeds were placed on the soil surface for each 9-cm-diameter pot, which was filled with a sterile potting mix containing peat, vermiculite, and soil (1:1:1 by v/v/v). A 1-cm-diameter mycelial plug taken from the margin of actively growing colonies (PDA) of the representative isolate KyrFa01 was contacted with each seed, and then seeds were covered with the same potting mix. The seeds in control pots were treated with sterile PDA plugs. The experiment was conducted in a growth chamber in a completely randomized design with five replicated pots at 23°C with a 12-h photoperiod. Disease assessment was made after 4 weeks of fungal inoculation. The isolate KyrFa01 induced discoloration on the crown and root tissues of inoculated plants similar to those observed in the field-grown plants, whereas no symptoms were observed on plants grown in the control pots. The pathogen was successfully reisolated from the symptomatic tissues, confirming Koch's postulates. To the best of our knowledge, this is the first report of crown rot caused by F. algeriense on wheat in Kyrgyzstan. Fusarium algeriense was firstly described within the Fusarium burgessii species complex by Laraba et al. (2017) as a crown rot pathogen of wheat in Algeria. The pathogen was secondly reported from wheat-growing areas in Azerbaijan (Özer et al. 2020a) and thirdly from Kyrgyzstan in this report. Özer et al. (2020b) confirmed the coexistence of this pathogen with other Fusarium species. The result warrants the need to further investigate the potential of this species in the Fusarium crown rot complex of wheat.

First Report of Crown Rot Caused by Fusarium redolens on Wheat in Kazakhstan.

Fusarium crown rot, caused by several species within the genus, is a major constraint that results in significant losses in wheat production worldwide. In June 2019, diseased wheat plants with typical symptoms of crown rot, including discoloration on the first two or three internodes of the stem just above the soil line and stunted, dry rotted, and discolored roots were collected in several bread wheat fields during the maturity stage in Almaty, East Kazakhstan, and Karaganda Regions of Kazakhstan. For each field, approximately twenty tillers were randomly sampled. Symptomatic tissues were surface sterilized in 1% NaClO for 2 min, rinsed with sterile distilled water three times, air-dried in a laminar flow hood, and then transferred to Petri dishes containing one-fifth strength potato dextrose agar (PDA). After incubating in the dark at 23°C for 5 days, 79 single-spore isolates showing cultural and microscopic characteristics of Fusarium were obtained on PDA and Spezieller-Nährstoffarmer agar (SNA). Colonies were initially white but later produced a beige to pink diffusible pigment in PDA. Microconidia that formed on aerial monophialides were hyaline, 0 to 1 septum, oval- to kidney-shaped, and measured 4.3 to 10.3 × 1.9 to 3.4 µm (average 7.8 × 2.6 µm), whilst macroconidia were straight to slightly curved, 3 to 5 septate, and measured 18.7 to 38.8 × 2.9 to 6.6 µm (average 29.9 × 4.7 µm), with foot-shaped basal cells on SNA. Chlamydospores were present on PDA. Sequence analysis based on portions of translation elongation factor 1α (TEF1) and the nuclear ribosomal internal transcribed spacer region (ITS rDNA) loci with primers EF1/EF2 (O'Donnell et al. 1998) and ITS1/ITS4 (White et al. 1990) identified 29 of the 79 isolates as Fusarium redolens Wollenw. The sequences of the five representative isolates with 99.85% of similarity to those of F. redolens strains available in GenBank e.g., ITS (MT435063) and TEF1 (GU250584). The TEF1 (accession nos. MW403914-MW403918) and ITS rDNA (accession nos. MW397138-MW397142) sequences of the isolates were deposited in GenBank. The morphological features are consistent with the described features of F. redolens (Leslie and Summerell 2006). To confirm pathogenicity of the five isolates, five pre-germinated seeds of wheat cultivar Seri 82 were placed in a 9-cm-diameter pot filled with a sterile potting mix containing equal volumes of peat, vermiculite, and soil. An approximately 1-cm-diameter 7-day-old mycelial plug of each isolate was individually placed in contact with the seeds. Seeds were covered with the same potting mix, and then the pots were maintained for four weeks in a growth chamber at 23°C with a 12-h photoperiod. The experiment was conducted twice with three replicate 15-cm pots with 5 plants per pot. Controls were inoculated with sterile agar plugs using the same procedure. After four weeks, all the inoculated plants showed stunted growth with brown discoloration in most parts of the crown and roots, whereas no symptoms were observed in the control plants. The mean severity of the disease for each isolate was between 2.1 and 2.7 according to the scale of 1 to 5 described by Gebremariam et al. (2015). The pathogen was reisolated from crowns of diseased plants, but not from asymptomatic control tissues, and identified morphologically based on the methods described above, fulfilling Koch's postulates. Although several morphological features are shared by F. oxysporum and F. redolens, Baayen et al. (2001) showed that these species could be easily distinguished using molecular data. The pathogen was previously reported as F. redolens associated with crown rot of wheat in Turkey (Gebremariam et al. 2015) and Saskatchewan, Canada (Taheri et al. 2011). The presence of F. redolens causing crown rot is confirmed in the six wheat fields surveyed in Kazakhstan, for the first time. This pathogen may pose a risk for wheat production, and further studies needed to determine the impact on the crop in Kazakhstan.

Plant-Parasitic Nematodes Associated With Wheat in Central, Eastern, and Southeastern Kazakhstan.

Kazakhstan is one of the biggest wheat producers, however, its wheat production is far below the average international wheat production standard due to biotic and abiotic stressors. Plant-parasitic nematodes are devastating for cereal production systems worldwide. A comprehensive survey was conducted in 2019 to identify plant-parasitic nematodes associated with wheat in different locations of central, eastern, and southeastern Kazakhstan. The results revealed 33 root-lesion and 27 cyst nematode populations from the 77 localities sampled. These two genera occurred in separate or in mixed populations. The root-lesion populations were identified as Pratylenchus neglectus and P. thornei while all cyst nematodes were identified as Heterodera filipjevi. The identification of nematodes was firstly performed based on morphological and morphometric features and confirmed by BLAST and phylogenetic analyses based on the internal transcribed spacer and the D2-D3 expansion located in the 28S gene of ribosomal DNA for CCN and RLN populations, respectively. Pratylenchus neglectus and P. thornei populations from Kazakhstan showed a high similarity with the American, European, and Asian populations. Heterodera filipjevi populations formed a well-supported cluster with the corresponding populations from different countries and showed a slightly intraspecific polymorphism. Kazakhstan populations of H. filipjevi may have multiple introductions in Kazakhstan due to the divergence among them. The results of this study are of great importance for breeding programs and will enable awareness for extension advisors to develop measures to control these nematodes in cereal cropping areas in Kazakhstan.

First Report of Rhizoctonia solani AG2-1 on roots of wheat in Kazakhstan.

In June 2019, approximately 20 tillers of wheat (Triticum aestivum L.) were sampled at the ripening stage (Feekes scale 11) from four different fields in Almaty, Kazakhstan. Brown lesions (3-5 mm in length) were present on the roots of sampled plants, with 20% incidence. To determine the causal agent, diseased roots were surface disinfected in sodium hypochlorite solution (1%) for 3 min, rinsed triple with sterile distilled water, air-dried in a laminar flow hood, and plated onto one-fifth strength potato dextrose agar (PDA) supplemented with 50 ppm chloramphenicol. After three days, the hyphal fragments that developed from the sections were transferred to fresh PDA and incubated at 23°C with 12-h photoperiod for 7 days to obtain pure cultures. Brown pigmented fungal colonies with a constriction at the base of hyphal branches, septa near the branching point, and right-angled branching resembling Rhizoctonia solani were observed. The identification anastomosis group (AG) of a representative isolate for each field was conducted by sequencing the internal transcribed spacer (ITS) region of rDNA with the universal primers ITS4 and ITS5 (White et al. 1990). The resulting sequences of 693 bp length were deposited in GenBank (accession nos. MW898143:MW898146). These sequences were 100% identical to the isolate 8Rs of R. solani AG2-1 (accession no. AF354063). To confirm the pathogenicity of the four isolates, the colonized wheat kernels method described by Demirci (1998) was used to inoculate a sterile potting mix containing peat, vermiculite, and soil (1:1:1 by v/v/v) into which wheat (cv. Seri) was planted. Control pots were inoculated with sterile wheat kernels using the same procedure. Wheat plants were left to grow for four weeks under controlled environmental conditions with a 23°C temperature regime. During the period that the plants remained in the glasshouse, the typical light regime was 16 h. Brown lesions were observed on the roots of plants in the inoculated pots whereas no symptoms were observed on plants grown in the control pots. R. solani was consistently reisolated from symptomatic plants, thereby confirming Koch's postulates. To our knowledge, this is the first report of R. solani AG2-1 on roots of wheat in Kazakhstan. R. solani AG2-1 isolates have been previously reported to be a weak pathogen to wheat (Roberts and Sivasithamparam 1986; Sturrock et al. 2015; Jaaffar et al. 2016; Özer et al. 2019). We suggest further studies are required to characterize the impact of R. solani AG2-1 in wheat. Considering crop rotation, the selection of non-host crops to this AG group is important to pathogen management, by reducing the amount of inoculum in the soil.

First Report of Fusarium culmorum and Microdochium bolleyi Causing Root Rot on Triticale in Kazakhstan.

Triticale (×Triticosecale Wittmack) is obtained from wheat × rye crossing. It is positioned between wheat and rye in terms of resistance to soilborne pathogens including Gaeumannomyces graminis var. tritici, Fusarium culmorum, F. avenaceum, and Bipolaris sorokiniana (Arseniuk and Góral 2015). In 2019, seven triticale fields were surveyed in Almaty Province, Kazakhstan to examine soil-borne fungal pathogens. A total of 28 symptomatic plants with stunting, rot or discolored root were collected to identify causal agents. The overall disease incidence was approximately 8 to 10% in the fields. Fungi were isolated from 3-5 mm pieces excised from symptomatic tissues. The pieces were exposed to surface disinfection in 1% sodium hypochlorite solution for 2 min, rinsed three times with sterile distilled water, blotted dry, and plated on 1/5 strength potato dextrose agar (PDA) amended with 0.01% streptomycin. Plates were left in the dark at 23°C for 7 days. A total of 34 fungal colonies were isolated of which nineteen isolates, originally from six fields showed the cultural characteristics of B. sorokiniana. This species was previously reported to cause common root rot on triticale in Kazakhstan (Özer et al. 2020). Ten isolates from four fields produced pale orange and cottony mycelium with red pigmentation on the agar, which is typical of Fusarium-like growth. The remaining isolates (n=5) from two fields produced salmon-colored and scarce aerial mycelium with no soluble pigmentation, similar to Microdochium spp. Fusarium isolates produced thick-walled and curved macroconidia with 3-4 septa (n=50, 25.7 to 37.6 × 4.1 to 7.3 µm in size) and notched basal cell on PDA, but microconidia were absent, which matches the description of F. culmorum (Wm.G. Sm.) Sacc. (Leslie and Summerell 2006). Microdochium isolates produced swollen, brown, and thick-walled chlamydospores and hyaline, one-celled, and thin-walled conidia (n=50, 5.4 to 9.3 × 1.5 to 3.0 µm in size) formed on ampullate and cylindrical conidiogenous cells on oatmeal agar (OA). These morphological features are consistent with previous observations for Microdochium bolleyi (R. Sprague) de Hoog & Herm.-Nijh. (Hong et al. 2008). To confirm morphological preliminary identifications, the portion of the translation elongation factor 1-alpha (EF1-α) gene was amplified with EF1/EF2 primers (O'Donnell et al. 1998) for representative Fusarium isolates (n=4) for each field. Additionally, the internal transcribed spacer (ITS) of ribosomal DNA was amplified with ITS1/ITS4 primers (White et al. 1990) for representative Microdochium isolates (n=2) for each field. BLASTn queries against NCBI GenBank revealed that the EF1-α sequences of Fusarium isolates (MW311081-MW311084) shared 100% identity with F. culmorum strain CBS 110262 (KT008433). The ITS sequences of M. bolleyi isolates (MW301448-MW301449) matched that of M. bolleyi strain CBS 137.64 (AM502264) with 100% sequence similarity. Pathogenicity test was conducted on pregerminated seeds of triticale cv. Balausa. A plastic pot (17 cm height, 9 cm in diam) was filled with a sterile mixture of vermiculite, peat, and soil (1:1:1, v/v/v). Mycelial plugs (1 cm in diam) were cut from the margin of a growing culture of representative isolates (Kaz_Fus123 and Kaz_Mb01) and placed onto the mixture in the pot. A sterile agar plug was employed as a control treatment. One pregerminated seed was put on the plug and covered with the mixture. The pots were transferred to a growth chamber set at 23 ± 2°C and a photoperiod of 14 hours. The experiment was performed twice using 5 replication pots per isolate. Four weeks after inoculation, discoloration of the crown was observed on all the inoculated roots, whereas no symptoms were observed on the control plants. Koch's postulates were fulfilled by reisolating and identifying the pathogen based on the morphology described above. This is the first report of M. bolleyi and F. culmorum causing root rot on triticale in Kazakhstan. Although B. sorokiniana is the most primary pathogen that may limit yield in the production of triticale in Kazakhstan, F. culmorum and M. bolleyi have been found to be less frequent and less aggressive pathogens, respectively. Further studies are needed to better understand the potential distribution and impact of these pathogens on triticale.

Reactive oxygen species metabolism and photosynthetic performance in leaves of Hordeum vulgare plants co-infested with Heterodera filipjevi and Aceria tosichella.

KEY MESSAGE: Defence responses of cyst nematode and/or wheat curl mite infested barley engage the altered reactive oxygen species production, antioxidant machinery, carbon dioxide assimilation and photosynthesis efficiency. The primary aim of this study was to determine how barley responds to two pests infesting separately or at once; thus barley was inoculated with Heterodera filipjevi (Madzhidov) Stelter (cereal cyst nematode; CCN) and Aceria tosichella Keifer (wheat curl mite; WCM). To verify hypothesis about the involvement of redox metabolism and photosynthesis in barley defence responses, biochemical, photosynthesis efficiency and chlorophyll a fluorescence measurements as well as transmission electron microscopy were implemented. Inoculation with WCM (apart from or with CCN) brought about a significant suppression in the efficiency of electron transport outside photosystem II reaction centres. This limitation was an effect of diminished pool of rapidly reducing plastoquinone and decreased total electron carriers. Infestation with WCM (apart from or with CCN) also significantly restricted the electron transport on the photosystem I acceptor side, therefore produced reactive oxygen species oxidized lipids in cells of WCM and double infested plants and proteins in cells of WCM-infested plants. The level of hydrogen peroxide was significantly decreased in double infested plants because of glutathione-ascorbate cycle involvement. The inhibition of nitrosoglutathione reductase promoted the accumulation of S-nitrosoglutathione increasing antioxidant capacity in cells of double infested plants. Moreover, enhanced arginase activity in WCM-infested plants could stimulate synthesis of polyamines participating in plant antioxidant response. Infestation with WCM (apart from or with CCN) significantly reduced the efficiency of carbon dioxide assimilation by barley leaves, whereas infection only with CCN expanded photosynthesis efficiency. These were accompanied with the ultrastructural changes in chloroplasts during CCN and WCM infestation.

Identity and Pathogenicity of Fungi Associated with Crown and Root Rot of Dryland Winter Wheat in Azerbaijan.

A comprehensive survey was performed to assess fungal populations associated with crown and root rot of wheat throughout the main wheat-growing areas of Azerbaijan. Samples were taken from 76 fields; 630 fungal strains were isolated, identified, and evaluated for pathogenicity. The identification was conducted with morphological and molecular tools such as species-specific PCR and DNA sequencing of the internal transcribed spacer (ITS) and translation elongation factor 1-α (EF1-α) loci. The fungus found in the greatest number of fields (44) was Fusarium culmorum with 192 isolates, followed by F. acuminatum. Other Fusarium spp. isolates were identified: F. equiseti, F. pseudograminearum, F. graminearum, F. incarnatum, F. avenaceum, F. hostae, F. oxysporum, F. proliferatum, F. algeriense, and F. brachygibbosum. Bipolaris sorokiniana, Curvularia spicifera, Exserohilum pedicellatum, Nigrospora oryzae, and Rhizoctonia spp. isolates were also identified, associated with underground parts of wheat. Phylogenetic analyses based on ITS and EF1-α sequences of the isolates showed that the isolates belonging to the same species were clearly separated in the dendrogram. Pathogenicity assays revealed that F. culmorum, F. pseudograminearum, and F. graminearum were most aggressive; F. avenaceum, F. hostae, F. algeriense, B. sorokiniana, C. spicifera, and R. solani isolates were moderately aggressive; C. inaequalis, E. pedicellatum, and N. oryzae were weakly aggressive; and others were nonpathogenic. The result of this study exhibited the existence of a wide range of species associated with crown and root rot of wheat in Azerbaijan. Additionally, this is the first report of F. hostae, F. algeriense, C. spicifera, C. inaequalis, and N. oryzae as pathogens on wheat in Azerbaijan. Azerbaijan is the second country after Algeria in which F. algeriense was detected.

Resistance to Cereal Cyst Nematodes in Wheat and Barley: An Emphasis on Classical and Modern Approaches.

Cereal cyst nematodes (CCNs) are among the most important nematode pests that limit production of small grain cereals like wheat and barley. These nematodes alone are estimated to reduce production of crops by 10% globally. This necessitates a huge enhancement of nematode resistance in cereal crops against CCNs. Nematode resistance in wheat and barley in combination with higher grain yields has been a preferential research area for cereal nematologists. This usually involved the targeted genetic exploitations through natural means of classical selection breeding of resistant genotypes and finding quantitative trait luci (QTLs) associated with resistance genes. These improvements were based on available genetic diversity among the crop plants. Recently, genome-wide association studies have widely been exploited to associate nematode resistance or susceptibility with particular regions of the genome. Use of biotechnological tools through the application of various transgenic strategies for enhancement of nematode resistance in various crop plants including wheat and barley had also been an important area of research. These modern approaches primarily include the use of gene silencing, exploitation of nematode effector genes, proteinase inhibitors, chemodisruptive peptides and a combination of one or more of these approaches. Furthermore, the perspective genome editing technologies including CRISPR-Cas9 could also be helpful for improving CCN resistance in wheat and barley. The information provided in this review will be helpful to enhance resistance against CCNs and will attract the attention of the scientific community towards this neglected area.

Genome-Wide Association Study for Multiple Biotic Stress Resistance in Synthetic Hexaploid Wheat.

Genetic resistance against biotic stress is a major goal in many wheat breeding programs. However, modern wheat cultivars have a limited genetic variation for disease and pest resistance and there is always a possibility of the evolution of new diseases and pests to overcome previously identified resistance genes. A total of 125 synthetic hexaploid wheats (SHWs; 2n = 6x = 42, AABBDD, Triticum aestivum L.) were characterized for resistance to fungal pathogens that cause wheat rusts (leaf; Puccinia triticina, stem; P. graminis f.sp. tritici, and stripe; P. striiformis f.sp. tritici) and crown rot (Fusarium spp.); cereal cyst nematode (Heterodera spp.); and Hessian fly (Mayetiola destructor). A wide range of genetic variation was observed among SHWs for multiple (two to five) biotic stresses and 17 SHWs that were resistant to more than two stresses. The genomic regions and potential candidate genes conferring resistance to these biotic stresses were identified from a genome-wide association study (GWAS). This GWAS study identified 124 significant marker-trait associations (MTAs) for multiple biotic stresses and 33 of these were found within genes. Furthermore, 16 of the 33 MTAs present within genes had annotations suggesting their potential role in disease resistance. These results will be valuable for pyramiding novel genes/genomic regions conferring resistance to multiple biotic stresses from SHWs into elite bread wheat cultivars and providing further insights on a wide range of stress resistance in wheat.

Resistance and tolerance reactions of winter wheat lines to Heterodera filipjevi in Turkey.

Nematodes attack cereal crops resulting in significant yield losses, estimated at 10%. The plant parasitic nematodes of the genus Heterodera attack cereals, particularly wheat, causing costly financial losses due to impact on yield. The soil borne pathogens program at the International Maize and Wheat Improvement Center (CIMMYT) in Turkey has focused on screening wheat germplasm to identify sources of Heterodera resistant varieties for almost 20 years. The aim of this current study was to validate the finding that resistant lines demonstrate resistant reactions under controlled conditions and to test whether they present tolerant reactions when challenged with cyst nematodes under two different locations in field conditions. The results of this study, including the check lines, indicated that 27 and 28 lines maintained their reactions to H. filipjevi in Eskisehir and Yozgat field, respectively, and 23 lines were the same in both locations. In terms of tolerance, 3 and 13 lines proven to be tolerant and moderately tolerant to H. filipjevi in Yozgat field. In Eskisehir field, 13 and 14 lines were tolerant and moderately tolerant. In both locations, L7 showed tolerance reaction, although it was susceptible. The majority of the resistant germplasm (60%, 14 lines) of screened lines from the Turkey CIMMYT-ICARDA (TCI) nursery were found to be resistant to both H. filipjevi populations including L1, L3, L6, L15, L21, L26, and L34, whereas 17% (four lines) from the USA had the same reaction. L32 showed a high level of resistance and tolerance in both locations and could prove to be promising lines in the breeding programs. The International Winter Wheat Improvement Program (IWWIP) formerly used these resistant lines in the crossing block and subsequently distributed them to more than 150 international collaborators. Regression analysis revealed a negative correlation between yield and RF of H. filipjevi in both nematode populations, which describes the negative impact of this pest on winter wheat. The results of this study are very important for breeding programs especially for the IWWIP, a joint program between the Turkish Ministry of Agriculture and Forestry, CIMMYT, and the International Centre for Agricultural Research in the Dry Areas (ICARDA).Nematodes attack cereal crops resulting in significant yield losses, estimated at 10%. The plant parasitic nematodes of the genus Heterodera attack cereals, particularly wheat, causing costly financial losses due to impact on yield. The soil borne pathogens program at the International Maize and Wheat Improvement Center (CIMMYT) in Turkey has focused on screening wheat germplasm to identify sources of Heterodera resistant varieties for almost 20 years. The aim of this current study was to validate the finding that resistant lines demonstrate resistant reactions under controlled conditions and to test whether they present tolerant reactions when challenged with cyst nematodes under two different locations in field conditions. The results of this study, including the check lines, indicated that 27 and 28 lines maintained their reactions to H. filipjevi in Eskisehir and Yozgat field, respectively, and 23 lines were the same in both locations. In terms of tolerance, 3 and 13 lines proven to be tolerant and moderately tolerant to H. filipjevi in Yozgat field. In Eskisehir field, 13 and 14 lines were tolerant and moderately tolerant. In both locations, L7 showed tolerance reaction, although it was susceptible. The majority of the resistant germplasm (60%, 14 lines) of screened lines from the Turkey CIMMYT­ICARDA (TCI) nursery were found to be resistant to both H. filipjevi populations including L1, L3, L6, L15, L21, L26, and L34, whereas 17% (four lines) from the USA had the same reaction. L32 showed a high level of resistance and tolerance in both locations and could prove to be promising lines in the breeding programs. The International Winter Wheat Improvement Program (IWWIP) formerly used these resistant lines in the crossing block and subsequently distributed them to more than 150 international collaborators. Regression analysis revealed a negative correlation between yield and RF of H. filipjevi in both nematode populations, which describes the negative impact of this pest on winter wheat. The results of this study are very important for breeding programs especially for the IWWIP, a joint program between the Turkish Ministry of Agriculture and Forestry, CIMMYT, and the International Centre for Agricultural Research in the Dry Areas (ICARDA).

Nematicidal Cyclic Lipodepsipeptides and a Xanthocillin Derivative from a Phaeosphariaceous Fungus Parasitizing Eggs of the Plant Parasitic Nematode Heterodera filipjevi.

The new cyclic lipodepsipeptide ophiotine (1), two new arthrichitin derivatives named arthrichitins B (4) and C (5), a new xanthocillin-like alkaloid, xanthomide Z (2), and the previously described arthrichitin (3) were isolated from the liquid culture broth of a nematode-associated fungus with affinities to the genus Ophiosphaerella. The structural elucidation and determination of the absolute configuration of the new molecules were accomplished using a combination of spectroscopic and chemical techniques, including 1D and 2D NMR, HRMS, and Marfey's analysis. Opiotine (1) displayed moderate nematicidal activity against the host nematode ( Heterodera filipjevi), while xanthomide Z (2) exhibited very weak activity. Arthrichitin C (5) showed very weak cytotoxic effects on several cancer cell lines, with IC50 values in the range of 24-33 µM. Xanthomide Z is among few xanthocillin derivatives that comprise formamide functions instead of the cyano functions that are usually observed in this class of fungal alkaloids.

Identification of Novel Quantitative Trait Loci Linked to Crown Rot Resistance in Spring Wheat.

Crown rot (CR), caused by various Fusarium species, is a major disease in many cereal-growing regions worldwide. Fusarium culmorum is one of the most important species, which can cause significant yield losses in wheat. A set of 126 advanced International Maize and Wheat Improvement Center (CIMMYT) spring bread wheat lines were phenotyped against CR for field crown, greenhouse crown and stem, and growth room crown resistance scores. Of these, 107 lines were genotyped using Diversity Array Technology (DArT) markers to identify quantitative trait loci linked to CR resistance by genome-wide association study. Results of the population structure analysis grouped the accessions into three sub-groups. Genome wide linkage disequilibrium was large and declined on average within 20 cM (centi-Morgan) in the panel. General linear model (GLM), mixed linear model (MLM), and naïve models were tested for each CR score and the best model was selected based on quarantine-quarantine plots. Three marker-trait associations (MTAs) were identified linked to CR resistance; two of these on chromosome 3B were associated with field crown scores, each explaining 11.4% of the phenotypic variation and the third MTA on chromosome 2D was associated with greenhouse stem score and explained 11.6% of the phenotypic variation. Together, these newly identified loci provide opportunity for wheat breeders to exploit in enhancing CR resistance via marker-assisted selection or deployment in genomic selection in wheat breeding programs.

Cereal Cyst Nematodes: A Complex and Destructive Group of Heterodera Species.

Small grain cereals have served as the basis for staple foods, beverages, and animal feed for thousands of years. Wheat, barley, oats, rye, triticale, rice, and others are rich in calories, proteins, carbohydrates, vitamins, and minerals. These cereals supply 20% of the calories consumed by people worldwide and are therefore a primary source of energy for humans and play a vital role in global food and nutrition security. Global production of small grains increased linearly from 1960 to 2005, and then began to decline. Further decline in production is projected to continue through 2050 while global demand for these grains is projected to increase by 1% per annum. Currently, wheat, barley, and oat production exceeds consumption in developed countries, while in developing countries the consumption rate is higher than production. An increasing demand for meat and livestock products is likely to compound the demand for cereals in developing countries. Current production levels and trends will not be sufficient to fulfill the projected global demand generated by increased populations. For wheat, global production will need to be increased by 60% to fulfill the estimated demand in 2050. Until recently, global wheat production increased mostly in response to development of improved cultivars and farming practices and technologies. Production is now limited by biotic and abiotic constraints, including diseases, nematodes, insect pests, weeds, and climate. Among these constraints, plant-parasitic nematodes alone are estimated to reduce production of all world crops by 10%. Cereal cyst nematodes (CCNs) are among the most important nematode pests that limit production of small grain cereals. Heavily invaded young plants are stunted and their lower leaves are often chlorotic, forming pale green patches in the field. Mature plants are also stunted, have a reduced number of tillers, and the roots are shallow and have a "bushy-knotted" appearance. CCNs comprise a number of closely-related species and are found in most regions where cereals are produced.

Genome-Wide Association Study in Wheat Identifies Resistance to the Cereal Cyst Nematode Heterodera filipjevi.

The cyst nematode Heterodera filipjevi is a plant parasite causing substantial yield loss in wheat. Resistant cultivars are the preferred method of controlling cyst nematodes. Association mapping is a powerful approach to detect associations between phenotypic variation and genetic polymorphisms; in this way favorable traits such as resistance to pathogens can be located. Therefore, a genome-wide association study of 161 winter wheat accessions was performed with a 90K iSelect single nucleotide polymorphism (SNP) chip. Population structure analysis grouped into two major subgroups and first principal component accounted 6.16% for phenotypic diversity. The genome-wide linkage disequilibrium across wheat was 3 cM. Eleven quantitative trait loci (QTLs) on chromosomes 1AL, 2AS, 2BL, 3AL, 3BL, 4AS, 4AL, 5BL, and 7BL were identified using a mixed linear model false discovery rate of P < 0.01 that explained 43% of total genetic variation. This is the first report of QTLs conferring resistance to H. filipjevi in wheat. Eight QTLs on chromosomes 1AL, 2AS, 2BL, 3AL, 4AL, and 5BL were linked to putative genes known to be involved in plant-pathogen interactions. Two other QTLs on 3BL and one QTL on 7BL linked to putative genes known to be involved in abiotic stress.

Association analysis of resistance to cereal cyst nematodes (Heterodera avenae) and root lesion nematodes (Pratylenchus neglectus and P. thornei) in CIMMYT advanced spring wheat lines for semi-arid conditions.

To identify loci linked to nematode resistance genes, a total of 126 of CIMMYT advanced spring wheat lines adapted to semi-arid conditions were screened for resistance to Heterodera avenae, Pratylenchus neglectus, and P. thornei, of which 107 lines were genotyped with 1,310 DArT. Association of DArT markers with nematode response was analyzed using the general linear model. Results showed that 11 markers were associated with resistance to H. avenae (pathotype Ha21), 25 markers with resistance to P. neglectus, and 9 significant markers were identified to be linked with resistance to P. thornei. In this work we confirmed that chromosome 4A (~90-105 cM) can be a source of resistance to P. thornei as has been recently reported. Other significant markers were also identified on chromosomal regions where no resistant genes have been reported for both nematodes species. These novel QTL were mapped to chromosomes 5A, 6A, and 7A for H. avenae; on chromosomes 1A, 1B, 3A, 3B, 6B, 7AS, and 7D for P. neglectus; and on chromosomes 1D, 2A, and 5B for P. thornei and represent potentially new loci linked to resistance that may be useful for selecting parents and deploying resistance into elite germplasm adapted to regions where nematodes are causing problem.

Genomic wide association study and selective sweep analysis identify genes associated with improved yield under drought in Turkish winter wheat germplasm.

A panel comprising of 84 Turkish winter wheat landraces (LR) and 73 modern varieties (MV) was analyzed with genome wide association study (GWAS) to identify genes/genomic regions associated with increased yield under favorable and drought conditions. In addition, selective sweep analysis was conducted to detect signatures of selection in the winter wheat genome driving the differentiation between LR and MV, to gather an understanding of genomic regions linked to adaptation and yield improvement. The panel was genotyped with 25 K wheat SNP array and phenotyped for agronomic traits for two growing seasons (2018 and 2019) in Konya, Turkey. Year 2018 was treated as drought environment due to very low precipitation prior to heading whereas year 2019 was considered as a favorable season. GWAS conducted with SNPs and haplotype blocks using mixed linear model identified 18 genomic regions in the vicinities of known genes i.e., TaERF3-3A, TaERF3-3B, DEP1-5A, FRIZZY PANICLE-2D, TaSnRK23-1A, TaAGL6-A, TaARF12-2A, TaARF12-2B, WAPO1, TaSPL16-7D, TaTGW6-A1, KAT-2B, TaOGT1, TaSPL21-6B, TaSBEIb, trs1/WFZP-A, TaCwi-A1-2A and TaPIN1-7A associated with grain yield (GY) and yield related traits. Haplotype-based GWAS identified five haplotype blocks (H1A-42, H2A-71, H4A-48, H7B-123 and H7B-124), with the favorable haplotypes showing a yield increase of > 700 kg/ha in the drought season. SNP-based GWAS, detected only one larger effect genomic region on chromosome 7B, in common with haplotype-based GWAS. On an average, the percentage variation (PV) explained by haplotypes was 8.0% higher than PV explained by SNPs for all the investigated traits. Selective sweep analysis detected 39 signatures of selection between LR and MV of which 15 were within proximity of known functional genes controlling flowering (PRR-A1, PPR-D1, TaHd1-6B), GY and GY components (TaSus2-2B, TaGS2-B1, AG1-1A/WAG1-1A, DUO-A1, DUO-B1, AG2-3A/WAG2-3A, TaLAX1, TaSnRK210-4A, FBP, TaLAX1, TaPIL1 and AP3-1-7A/WPA3-7A) and 10 regions underlying various transcription factors and regulatory genes. The study outcomes contribute to utilization of LR in breeding winter wheat.

Xenorhabdus and Photorhabdus Bacteria as Potential Candidates for the Control of Culex pipiens L. (Diptera: Culicidae), the Principal Vector of West Nile Virus and Lymphatic Filariasis.

Vector-borne diseases pose a severe threat to human and animal health. Culex pipiens L. (Diptera: Culicidae) is a widespread mosquito species and serves as a vector for the transmission of infectious diseases such as West Nile disease and Lymphatic Filariasis. Synthetic insecticides have been the prime control method for many years to suppress Cx. pipiens populations. However, recently, the use of insecticides has begun to be questioned due to the detrimental impact on human health and the natural environment. Therefore, many authorities urge the development of eco-friendly control methods that are nontoxic to humans. The bacterial associates [Xenorhabdus and Photorhabdus spp. (Enterobacterales: Morganellaceae)] of entomopathogenic nematodes (EPNs) (Sterinernema spp. and Heterorhabditis spp.) (Rhabditida: Heterorhabditidae and Steinernematidae) are one of the green approaches to combat a variety of insect pests. In the present study, the mosquitocidal activity of the cell-free supernatants and cell suspension (4 × 107 cells mL-1) of four different symbiotic bacteria (Xenorhabdus nematophila, X. bovienii, X. budapestensis, and P. luminescens subsp. kayaii) was assessed against different development stages of Cx. pipiens (The 1st/2nd and 3rd/4th instar larvae and pupa) under laboratory conditions. The bacterial symbionts were able to kill all the development stages with varying levels of mortality. The 1st/2nd instar larvae exhibited the highest susceptibility to the cell-free supernatants and cell suspensions of symbiotic bacteria and the efficacy of the cell-free supernatants and cell suspensions gradually declined with increasing phases of growth. The highest effectiveness was achieved by the X. bovienii KCS-4S strain inducing 95% mortality to the 1st/2nd instar larvae. The results indicate that tested bacterial symbionts have great potential as an eco-friendly alternative to insecticides.

Evaluation of Entomopathogenic Nematodes against Common Wireworm Species in Potato Cultivation.

Wireworms (Coleoptera: Elateridae) are common insect pests that attack a wide range of economically important crops including potatoes. The control of wireworms is of prime importance in potato production due to the potential damage of the larvae to tuber quantity and quality. Chemical insecticides, the main control strategy against wireworms, generally fail to provide satisfactory control due to the lack of available chemicals and the soil-dwelling habits of the larvae. In the last decades, new eco-friendly concepts have emerged in the sustainable control of wireworms, one of which is entomopathogenic nematodes (EPNs). EPNs are soil-inhabitant organisms and represent an ecological approach to controlling a great variety of soil-dwelling insect pests. In this study, the susceptibility of Agriotes sputator Linnaeus and A. rufipalpis Brullé larvae, the most common wireworm species in potato cultivation in Türkiye, to native EPN strains [Steinernema carpocapsae (Sc_BL22), S. feltiae (Sf_BL24 and Sf_KAY4), and Heterorhabditis bacteriophora (Hb_KAY10 and Hb_AF12)] were evaluated at two temperatures (25 and 30 °C) in pot experiments. Heterorhabditis bacteriophora Hb_AF12 was the most effective strain at 30 °C six days post-inoculation and caused 37.5% mortality to A. rufipalpis larvae. Agriotes sputator larvae were more susceptible to tested EPNs at the same exposure time, and 50% mortality was achieved by two EPNs species, Hb_AF12 and Sc_BL22. All EPN species/strains induced mortality over 70% to both wireworm species at both temperatures at 100 IJs/cm2, 18 days post-treatment. The results suggest that tested EPN species/strains have great potential in the control of A. sputator and A. rufipalpis larvae.

Fungal Pathogens Associated with Crown and Root Rot in Wheat-Growing Areas of Northern Kyrgyzstan.

Fungal species associated with crown and root rot diseases in wheat have been extensively studied in many parts of the world. However, no reports on the relative importance and distribution of pathogens associated with wheat crown and root rot in Kyrgyzstan have been published. Hence, fungal species associated with wheat crown/root rot were surveyed in three main wheat production regions in northern Kyrgyzstan. Fungal species were isolated on 1/5 strength potato-dextrose agar amended with streptomycin (0.1 g/L) and chloramphenicol (0.05 g/L). A total of 598 fungal isolates from symptomatic tissues were identified using morphological features of the cultures and conidia, as well as sequence analysis of the nuclear ribosomal internal transcribed spacer (ITS) region, the translation elongation factor 1α (TEF1), and the RNA polymerase II beta subunit (RPB2) genes. The percentage of fields from which each fungus was isolated and their relative percentage isolation levels were determined. Bipolaris sorokiniana, the causal agent of common root rot, was the most prevalent pathogenic species isolated, being isolated from 86.67% of the fields surveyed at a frequency of isolation of 40.64%. Fusarium spp. accounted for 53.01% of all isolates and consisted of 12 different species. The most common Fusarium species identified was Fusarium acuminatum, which was isolated from 70% of the sites surveyed with an isolation frequency of 21.57%, followed by Fusarium culmorum, Fusarium nygamai, Fusarium oxysporum, and Fusarium equiseti, all of which had a field incidence of more than 23%. Inoculation tests with 44 isolates representing 17 species on the susceptible Triticum aestivum cv. Seri 82 revealed that Fusarium pseudograminearum and F. culmorum isolates were equally the most virulent pathogens. The widespread distribution of moderately virulent B. sorokiniana appears to be a serious threat to wheat culture, limiting yield and quality. With the exception of F. culmorum, the remaining Fusarium species did not pose a significant threat to wheat production in the surveyed areas because common species, such as F. acuminatum, F. nygamai, F. oxysporum, and F. equiseti, were non-pathogenic but infrequent species, such as Fusarium redolens, Fusarium algeriense, and F. pseudograminearum, were highly or moderately virulent. Curvularia inaequalis, which was found in three different fields, was mildly virulent. The remaining Fusarium species, Fusarium solani, Fusarium proliferatum, Fusarium burgessii, and Fusarium tricinctum, as well as Microdochium bolleyi, Microdochium nivale, and Macrophomina phaseolina, were non-pathogenic and considered to be secondary colonizers. The implications of these findings are discussed.

Polydomus karssenii gen. nov. sp. nov. is a dark septate endophyte with a bifunctional lifestyle parasitising eggs of plant parasitic cyst nematodes (Heterodera spp.).

In this study fungal strains were investigated, which had been isolated from eggs of the cereal cyst nematode Heterodera filipjevi, and roots of Microthlaspi perfoliatum (Brassicaceae). The morphology, the interaction with nematodes and plants and the phylogenetic relationships of these strains originating from a broad geographic range covering Western Europe to Asia Minor were studied. Phylogenetic analyses using five genomic loci including ITSrDNA, LSUrDNA, SSUrDNA, rpb2 and tef1-α were carried out. The strains were found to represent a distinct phylogenetic lineage most closely related to Equiseticola and Ophiosphaerella, and Polydomus karssenii (Phaeosphaeriaceae, Pleosporales) is introduced here as a new species representing a monotypic genus. The pathogenicity tests against nematode eggs fulfilled Koch's postulates using in vitro nematode bioassays and showed that the fungus could parasitise its original nematode host H. filipjevi as well as the sugar beet cyst nematode H. schachtii, and colonise cysts and eggs of its hosts by forming highly melanised moniliform hyphae. Light microscopic observations on fungus-root interactions in an axenic system revealed the capacity of the same fungal strain to colonise the roots of wheat and produce melanised hyphae and microsclerotia-like structure typical for dark septate endophytes. Confocal laser scanning microscopy further demonstrated that the fungus colonised the root cells by predominant intercellular growth of hyphae, and frequent formation of appressorium-like as well as penetration peg-like structures through internal cell walls surrounded by callosic papilla-like structures. Different strains of the new fungus produced a nearly identical set of secondary metabolites with various biological activities including nematicidal effects irrespective of their origin from plants or nematodes.