Nitrogen reduction combined with ETc irrigation maintained summer maize yield and increased water and nitrogen use efficiency.

Introduction: High rainfall and excessive urea application are counterproductive to summer maize growth requirements and lower grain yield and water/nitrogen (N) use efficiency. The objective of this study was to determine whether ETc irrigation based on summer maize demand and reduced nitrogen rate in the Huang Huai Hai Plain increased water and nitrogen use efficiency without sacrificing yield. Methods: To achieve this, we conducted an experiment with four irrigation levels [ambient rainfall (I0) and 50% (I1), 75% (I2), and 100% (I3) of actual crop evapotranspiration (ETc)] and four nitrogen rates [no nitrogen fertilizer (N0), recommended nitrogen rate of urea (NU), recommended nitrogen rate of blending controlled-release urea with conventional urea fertilizer (BCRF) (NC), and reduced nitrogen rate of BCRF (NR)] in 2016-2018. Results: The results show that reduced irrigation and nitrogen rate reduced Fv/Fm, 13C-photosynthate, and nitrogen accumulation both in the kernel and plant. I3NC and I3NU accumulated higher 13C-photosynthate, nitrogen, and dry matter. However, 13C-photosynthate and nitrogen distribution to the kernel was decreased from I2 to I3 and was higher in BCRF than in urea. I2NC and I2NR promoted their distribution to the kernel, resulting in a higher harvest index. Compared with I3NU, I2NR increased root length density by 32.8% on average, maintaining considerable leaf Fv/Fm and obtaining similar kernel number and kernel weight. The higher root length density of I2NR of 40-60 cm promoted 13C-photosynthate and nitrogen distribution to the kernel and increased the harvest index. As a result, the water use efficiency (WUE) and nitrogen agronomic use efficiency (NAUE) in I2NR increased by 20.5%-31.9% and 11.0%-38.0% than that in I3NU, respectively. Discussion: Therefore, 75%ETc deficit irrigation and BCRF fertilizer with 80% nitrogen rate improved root length density, maintained leaf Fv/Fm in the milking stage, promoted 13C-photosynthate, and distributed nitrogen to the kernel, ultimately providing a higher WUE and NAUE without significantly reducing grain yield.

Comparative transcriptomic reveals the molecular mechanism of maize hybrid Zhengdan538 in response to water deficit.

Heterosis, known as one of the most successful strategies for increasing grain yield and abiotic/biotic stress tolerance, has been widely exploited in maize breeding. However, the underlying molecular processes are still to be elucidated. The maize hybrid "Zhengdan538" shows high tolerance to drought stress. The transcriptomes of the seedling leaves of its parents, "ZhengA88" and "ZhengT22" and their reciprocal F1 hybrid under well-watered and water deficit conditions, were analyzed by RNA sequencing (RNA-Seq). Transcriptome profiling of the reciprocal hybrid revealed 2994-4692 differentially expressed genes (DEGs) under well-watered and water-deficit conditions, which were identified by comparing with their parents. The reciprocal hybrid was more closely related to the parental line "ZhengT22" than to the parental line "ZhengA88" in terms of gene expression patterns under water-deficit condition. Furthermore, genes showed expression level dominance (ELD), especially the high-parental ELD (Class 3 and 5), accounted for the largest proportion of DEGs between the reciprocal F1 hybrid and their parental lines under water deficit. These ELD genes mainly participated in photosynthesis, energy biosynthesis, and metabolism processes. The results indicated that ELD genes played important roles in hybrid tolerance to water deficit. Moreover, a set of important drought-responsive transcription factors were found to be encoded by the identified ELD genes and are thought to function in improving drought tolerance in maize hybrid plants. Our results provide a better understanding of the molecular mechanism of drought tolerance in hybrid maize.

Molecular mapping of quantitative trait loci for 3 husk traits using genotyping by sequencing in maize (Zea mays L.).

The maize (Zea mays L.) husk consists of multiple leaf layers and plays an important role in grain growth and development. Despite significant achievements in physiological and morphological research, few studies have focused on the detection of genetic loci underlying husk-related traits due to the lack of efficient tools. In this study, we constructed an ultra-high-density linkage map using genotyping by sequencing based on a recombinant inbred line population to estimate the genetic variance and heritability of 3 husk traits, i.e. husk length, husk width, and husk layer number in 3 field environments and the combined environment. The 3 husk traits showed broad phenotypic variation and high heritability; the broad-sense heritability (H2) was 0.92, 0.84, and 0.86. Twenty quantitative trait loci were consistently detected more than 1 environment, including 9 for husk length, 6 for husk width, and 5 for husk layer number. These loci were considered as stable quantitative trait loci. Based on the quantitative trait loci mapping in the recombinant inbred line population, qHL6 and qHN4 were detected across all environments and inferred to be reliable and major-effect quantitative trait loci for husk length and husk layer number, respectively. In addition, several predicted candidate genes were identified in the region of qHL6 and qHN4, of which 17 candidate genes potentially play a role in biological processes related to development process and energy metabolism. These results will be as a useful resource for performing functional studies aimed at understanding the molecular pathways involved in husk growth and development.

Bulk analysis by resequencing and RNA-seq identifies candidate genes for maintaining leaf water content under water deficit in maize.

Drought is one of the main abiotic stresses adversely affecting maize growth and grain yield. Identifying drought tolerance-related genes and breeding varieties with enhanced tolerance are effective strategies for minimizing the effects of drought stress. In this study, the leaf relative water content (LRWC) was used for evaluating drought tolerance. QTL-seq analysis of 419 F2 individuals from a cross between ZhengT22 (the drought-tolerant line with high LRWC) and ZhengA88 (the drought-sensitive line with low LRWC) revealed four LRWC-related QTLs (qLRWC2, qLRWC10a, qLRWC10b, and qLRWC10c) in maize seedlings under water deficit. Of these QTLs, qLRWC2 was located in a 2.03-Mb interval on chromosome 2, whereas qLRWC10a, qLRWC10b, and qLRWC10c were located in 2.85-, 3.99-, and 2.05-Mb intervals, respectively, on chromosome 10, and the 93 genes contained the variation loci locating in the four QTLs regions. To identify the candidate genes within the QTLs, an RNA-seq analysis was performed for the parents exposed to water deficit. Seven genes with effective variation loci showed significant difference in expression either in ZhengA88 or ZhengT22 in response to water deficit. Moreover, among the genes, ZmPrx64, ZmCIPK, HSP90, and ABCG34 have all been shown to be related to water stress in the previous studies. Thus, they are primary considered as the potential candidate genes controlling LRWC under water deficit at the seeding stage of maize in this study. These findings will help clarify the molecular basis of drought tolerance in maize seedlings and may be relevant for future functional analysis and for breeding drought-tolerant maize varieties.

First Report of Maize Stalk Rot Caused by Fusarium kyushuense in China.

During 2017 to 2019, a field survey for maize stalk rot was conducted in 21 counties (districts) across the Guangxi province of China. This disease caused yield losses ranging from 20% to 30%. Maize plants with stalk rot were collected during the late milk stage and pieces of diseased pith tissue were cultured as previously described (Shan et al. 2017). Fungal colonies and mycelia with morphological characteristics of Fusarium species were subcultured onto fresh potato dextrose agar (PDA) and carnation leaf agar (CLA) plates. Based on morphological characteristics and molecular detection by amplification of Fusarium genus-specific primers (Duan et al. 2016), 39 Fusarium isolates were identified. Among them, five isolates from Du'an, Pingguo, Debao, and Daxin had abundant, pale orange to yellow aerial mycelium with deep red pigments when grown on PDA (Fig. 1A; 1B). The average growth rate was 8.0 to 12.0 mm per day at 25°C in the dark. The fungi produced two types of spores on CLA. Microconidia were ovoid to clavate, generally 0- to 3-septate, and 4.6 to 9.4 µm in length (n = 30) (Fig. 1D); Macroconidia were slightly curved with an acute apical cell, mostly 3- to 4- septate, and 19.4 to 38.2 µm in length (n = 30) (Fig. 1C). No chlamydospores were observed. These five isolates were initially identified as Fusarium kyushuense based on morphological features. PCR was performed to amplify three phylogenetic genes (TEF1-α, RPB1, and RPB2) (O'Donnell et al. 1998) and species specific primers kyuR1F/kyuR1R (5-TTTTCCTCACCAAGGAGCAGATCATG-3/5-TCCAATGGACTGGGCAGCCAAAACACC-3), kyuR2F/kyuR2R (5-CAGATATACATTTGCCTCGACAC-3/5-TACTTGAGCACGGAGCTTG-3) were used to confirm species identity. The obtained sequences were deposited in GenBank under the accession numbers MT997084, MT997080, MT997081 (TEF1-α); MT550012, MT997085, MT997086 (RPB1); MT550009, MT997089, and MT997090 (RPB2), respectively. Using BLAST, sequences of TEF1-α, RPB1, and RPB2 of the isolates were 99.33% (MH582297.1) to 100% (MG282364.1) similar to those of F. kyushuense strains (Supplementary Table 1). Based on phylogenetic analysis with maximum likelihood methods using tools of the website of CIPRES (http://www.phylo.org), isolates GX27, GX167, and GX204 clustered with F. kyushuense with 100% bootstrap support (Fig. 2). The pathogenicity of the three isolates was tested using young seedlings and adult plants as previously described with modification (Ye et al. 2013; Zhang et al. 2016). The primary roots of three-leaf-old seedlings were inoculated by immersing the roots into a 1 × 106 macroconidia solution, incubating for 6 h at 25°C, and transferring to normal growth conditions (26°C, 16 h light/22°C, 8 h dark). The second or third internode above the soil surface of flowering stage plants grown in a greenhouse was bored with a Bosch electric drill to make a hole (ca. 8 mm in diameter) and inoculated with 0.5 mL of mycelia plug then sealed with petrolatum. The inoculum was created by homogenizing five plates of flourish hyphal mats (approximately 125 mL) with kitchen blender and adjusting to a final volume of 200 mL with sterilized ddH2O. No symptoms were observed in the seedlings or adult plants that were mock-inoculated with PDA plugs. Three days post-inoculation (dpi), roots of the infected seedling turned dark-brown and shrunk and the leaves wilted (Fig. 1E). Typical stalk rot symptoms observed in the inoculated plants were premature wilting of entire plant and hollow and weak stalks, leading to lodging; the longitudinal section of the internodes exhibited obvious dark brown necrosis and reddish discoloration at 14 dpi and 30 dpi, respectively (Fig. 1F). Fusarium kyushuense was re-isolated from the inoculated stalk lesions but not from the control. This is the first record of stalk rot caused by F. kyushuense on maize plants in China. However, F. kyushuense is known to cause maize ear rot in China (Wang et al. 2014) and can produce type A and type B trichothecene mycotoxins in kernels (Aoki and O'Donnell 1998). The occurrence of maize stalk rot and ear rot caused by F. kyushuense should be monitored in China due to the potential risk for crop loss and mycotoxin contamination.

First Report of Fusarium thapsinum Causing Maize Stalk Rot in China.

Maize (Zea mays L.) is the most widely grown crop in China, which was planted 41.28 million hectares in 2019 (http://data.stats.gov.cnw/easyquery.htm?cn=C01&zb=A0D0F&sj=2019). Several fungal diseases of maize are reported in which stalk rot has become one of the most destructive diseases in China. The average yield losses affected by the disease are estimated at 10% to 20% (Yu et al. 2016). From 2017 to 2019, a survey was conducted to determine the population diversity of Fusarium species associated with maize diseases in 18 cities across Henan province. Fusarium stalk rot of maize with disease incidence more than 25% was observed in two continuous maize fields at Xuchang city. The diseased stem tissues from junctions in health and disease were chopped into small pieces (3 × 8 mm), superficially disinfected (70% ethyl alcohol for 1 min), placed onto potato dextrose agar (PDA) amended with L-(+)-Lactic-acid (1 g/L), poured in petri plates and incubated at 25°C for 4 days. Mycelia showing morphological characteristic of Fusarium spp. were sub-cultured from single conidium. The pure fungal isolates produced fluffy colonies, white aerial mycelium with yellow pigment in agar. The radial mycelial growth was measured and calculated at an average growth rate 10.9 mm/day at 25°C (Fig. 1A; 1B). Macroconidia produced on carnation leaf agar (CLA) were relatively slender, slightly curved and thick-walled, mostly 3 to 5 marked septa, with a curved and tapering apical cell and poorly developed foot cell, 46.9 ± 5.6 µm × 4.9 ± 0.2 µm (Fig. 1C). Microconidia formed abundantly and were generally oval on CLA, 8.2 ± 0.5 µm × 3.4± 0.1 µm (Fig. 1D). No chlamydospores were observed. Morphological characteristics of the isolates matched the description of Fusarium thapsinum (Leslie and Summerell 2006). To further get the phylogenetic evidence, TEF1-α (translation elongation factor), RPB1 (the largest subunit of RNA polymerase II) and RPB2 (the second largest subunit of RNA polymerase II) were amplified with primer pairs EF1/EF2 (O'Donnell et al. 1998), thapR1F (5'-TTTTCCTCACAAAGGAGCAAATCATG-3')/thapR1R (5'-GTTCACCCAAGATATGGTCGAAAGCC-3'), and thapR2F (5'-ACTCTTTCACATTTGCGCCGAAC-3')/thapR2R (5'-CGGAGCTTTCGTCCAGTGTGAC-3'), and sequenced, respectively. The BLAST search of the sequences of EF1-α, RPB1 and RPB2 shared 99.87% to 100% identity with those of F. thapsinum strains deposited in the GenBank (Supplementary Table 1). Sequences from two isolates (XCCG-3-B-1 and XCCG-3-A-1) were deposited in GenBank (Accession No. MT550014, MT997082 for EF-1α; MT550011, MT997087 for RPB1 and MT550008, MT997091 for RPB2). The phylogenetic relationships based on analysis of the partial sequences showed the representive isolates clustered together with F. thapsinum at 96% bootstrap values (Fig. 2). Combined with the results of morphological characteristics and phylogenetic analysis, the strain designated as Fusarium thapsinum. To complete Koch's postulates, the pathogenicity of the isolates was tested using the silking-stage plants in a greenhouse based on previously described method with modification (Zhang et al. 2016). An 8 mm in diameter wound hole was created at the second or third internode of the plant above the soil surface and injected with 0.5 ml of mycelia plug. The inoculated stalk exhibited internal dark brown necrotic regions and the brown area elongated obviously around the insertion at 14 dpi (days post inoculation). At 30 dpi, the stalks turned soft, hollow and even lodging of the plants for those severe ones, which are similar to those observed on naturally infected maize plants in the field (Fig. 1F). When the roots of the three-leaf-stage seedlings were inoculated with 1×106 macroconidia solution (Ye et al. 2013), the root rot and leaf wilting symptoms were observed (Fig. 1E). While the control plants that were inoculated with only sterile water showed no disease symptoms. The pathogen was re-isolated from the inoculated tissues and the identity was confirmed by the morphological characters. Fusarium thapsinum had been described as causal agent of maize stalk rot in Pakistan (Tahir et al. 2018). To our knowledge, this is the first report of F. thapsinum associated with maize stalk rot in China. The discovery will strengthen the theoretical foundation of maize stalk rot disease management.

Molecular mapping of quantitative trait loci for grain moisture at harvest and field grain drying rate in maize (Zea mays L.).

Maize (Zea mays L.) grain moisture (GM) at harvest is an important trait that affects seed preservation during storage, grain quality and artificial drying costs. To date, most of the work on understanding GM dynamics in maize has focused on the grain filling period, while the period of postmaturity grain drying remains unexplored. The field grain drying rate (FDR) is one of the most important factors in determining GM at harvest. Therefore, understanding the genetic basis of FDR will be useful for obtaining low-GM varieties. In this study, a single-cross population (330 F2:3 -generation plants) derived from a cross of two divergent inbred lines was evaluated in two planting environments with a measurement method - Area under the Dry Down Curve (AUDDC). A high-density genetic linkage map of 2491 single nucleotide polymorphism (SNP) loci covering 2415.56 cM was constructed. Using composite interval mapping, four quantitative trait loci (QTL), q45dGM1-1, qHTGM2-2, qAUDDC2-1 and qAUDDC10-1, which were detected on chromosomes 1, 2 and 10, were stable across environments and could explain more than 10% of phenotypic variance. These may be the major QTLs, with non-significant environmental interactions for GM at 45 days, GM at harvest and FDR, respectively. Additionally, several predicted candidate genes for FDR were identified, including several transcription factors, hormone responsive genes, energy-related and DNA replication-related genes. These results will provide useful information for our understanding of the genetic basis of FDR, as well as providing tools for marker-assisted selection in maize breeding.