Pathogenic aspects of Pantoea agglomerans in relation to cotton boll age and Dysdercus cingulatus (Fabricius) transmitting seed and boll rot in cotton germplasm

Bacterial seed and boll rot is a newly emerging cotton disease in Pakistan. Twenty-one cotton varieties were screened to find resistance source against the disease. None of these was found to be resistant. Five cotton varieties (CIM-595, MK2, BT-986, BT-986 & SG-1) having 700–1400 Area under disease progress curve (AUDPC) units were found to be moderately resistant to the disease. SLH-317, FH-942, BT-222, BT-666, MNH-457 ranging from 1401–1700 AUDPC units were moderately susceptible while MNH-456, SLH-336, 9811, FH-942, MNH-886 susceptible to boll rot. Seven varieties (FH-114, FH-113, BT-7, BT-212, SLH-BT-4, BT-212 and FH-941) were highly susceptible to bacterial seed and boll rot indicated by 2001–2300 AUDPC units. Biochemical tests identified bacterial isolates as Pantoea agglomerans. Different inoculation techniques were assessed for bacterial pathogenicity and symptoms of boll rot were only observed in needle punctured bolls. One, two and three weeks old bolls were mechanically inoculated by injecting bacterial suspension to evaluate the boll’s age impact on disease severity. Maximum severity was observed in two weeks old bolls. Red cotton bugs (Dysdercus cingulatus) were fed on artificially inoculated diseased bolls and then transferred on healthy bolls. Diseased symptoms were noticed on healthy cotton bolls. Bacterial colonies were recovered and red cotton bug was confirmed as the disease-transmitting vector.     

Fungi associated with boll and lint rot of cotton in Southern Khorasan province of Iran

During surveys of cotton fields since 2009 in the Southern Khorasan province of Iran, symptomatic samples with lint and boll rot disease signs were collected and cultured on potato dextrose, malt extract agar and PARPH media. Several fungal isolates were obtained and identified. Among them, we found Penicillium expansum which to our knowledge is reported as a new pathogen of cotton worldwide; Fusarium semitectum and Nigrospora oryzae (associated to mite Siteroptes sp.) were also recorded as lint or boll rot pathogens for the first time from Iran. Disease symptoms and fungal characteristics of Exserohilum rostratum, which has been reported briefly as a new causal agent of boll and lint rot disease, are illustrated and presented. In addition, Aspergillus niger and Rhizopus spp. were also isolated frequently from lint parts of cotton prior to harvesting. There was a positive correlation between disease occurrence and spiny cotton bollworm damage. The pathogenicity test was characterised by inoculating the attached and detached bolls of cotton plants.     

Role of Pantoea agglomerans in opportunistic bacterial seed and boll rot of cotton (Gossypium hirsutum) grown in the field

AIMS: To investigate the aetiology of seed and boll rot of cotton grown in South Carolina (SC). METHODS AND RESULTS: Bacteria were isolated from diseased locules of cotton bolls collected in a field in SC, USA and tested for the ability to cause comparable disease symptoms in greenhouse grown cotton fruit. Spontaneously generated rifampicin-resistant (Rif(r)) mutants of the isolates were used in confirmatory pathogenicity tests. Resistance to the antibiotic was both stable and effective in differentiating between an inoculated Rif(r) strain, rifampicin-sensitive contaminants and/or endophytes. A series of inoculation methods was tested at various boll developmental stages and at different fruiting nodes on the plant. Field disease symptoms were reproduced by inoculating bolls at 2 weeks postanthesis with bacterial suspensions ranging from 10(3) to 10(6) CFU ml(-1). Pathogenic isolates were categorized as Pantoea agglomerans on the basis of phenotype testing, fatty acid profiling (similarity index = 0.94), and 16s ribosomal DNA sequence analysis (99% nucleotide identity). CONCLUSIONS: Pantoea agglomerans isolates from field-collected immature, diseased cotton caused comparable infection symptoms in greenhouse produced cotton fruit. SIGNIFICANCE AND IMPACT OF THE STUDY: In 1999, significant yield losses in SC cotton resulted from a previously unobserved seed and boll rot that has since been reported in other southeastern states. This study demonstrated a role of P. agglomerans in producing opportunistic bacterial seed and boll rot of cotton.     

Natural deuterium and oxygen-18 enrichment in leaf water of cotton plants grown under wet and dry conditions: evidence for water compartmentation and its dynamics

Abstract. Significant differences in leaf water oxygen and hydrogen isotopic composition were observed between cotton plants grown under wet and dry conditions. The magnitude of the differences could be fully explained by the conventional model that describes the isotopic composition of an evaporating water pool under steady state conditions. The results indicate that leaf water isotopic composition is strongly influenced by transpiration rate via its effects on relative humidity adjacent to the leaf surface and on the isotopic composition of the air moisture. Our application of the model, however, provides evidence that leaf water must consist of a mixture of several isotopically distinct pools. These pools are suggested to reside in the symplast, in the cell walls and intercellular spaces and in the veins. A model is proposed suggesting that only the water residing in the cell walls and the intercellular spaces (the transpiration pool) interacts directly with the external environment. The large symplastic pool responds to the external environment to a limited extent via its relatively slow exchange with water in the transpiration pool. It is likely that the isotopic composition of water in the symplastic pool is strongly buffered against shortterm environmental variations, a possibility that would have important implications for the isotopic conditions under which organic matter biosynthesis occurs.     

Ecological impact on development of hemipterous bug (dysdercus koenigii) (hemiptera: pyrrhocoridae) and boll rot disease of cotton (gossypium hirsutum) grown in the diversified field

The experiment was conducted at Cotton Research Station, Multan to study the impact of weather factors and Hemipterous bug on development of cotton boll disease in cotton variety bt- 886 for three consecutive years i.e., 2012, 2013 and 2014. The results revealed that the population of Red Cotton Bug (RCB) per plant remain 0.50 and 0.34 during years 2012 and 2013, respectively but increased during 2014 i.e., 3.21 per plant. The number of unopened bolls (UOB) were more during 2012 i.e., 13.43% with yellowish lint (YL) 76.30% and whitish lint (WL) 23.70% at average maximum temperature of 34.73◦C, minimum temperature of 22.83◦C, RH of 77.43% and 11.08 mm rainfall. Similarly during 2013, the number of unopened bolls were less i.e., 0.34 per plant with YL 1.48 and WL 99.53 per plant when average maximum temperature 34.60^◦C, minimum temperature 23.37^◦C, RH 73.01% and 9.95 mm rainfall. During 2014, RCB population per plant was 3.22 with no UOB and YL was 0.00% and WL was 100% when average maximum temperature 23.70^◦C, minimum temperature 23.18◦C, RH 71.67% and 4.55 mm rainfall. So our results concluded that the cotton bolls rot disease was more during 2012 due to abrupt changes in environmental factors. The RCB may be the carrier of boll rot disease pathogen during more rainfall.     

棉花萌发期和苗期耐盐性评价及耐盐指标筛选

于2010年在江苏南京农业大学人工气候室内进行砂培试验,采用不同浓度的NaCl水溶液模拟盐胁迫,以多项指标盐害系数隶属函数值和总隶属函数值为依据,比较了13个棉花品种萌发期和苗期的耐盐性,并进行聚类分析.结果表明:150mmol·L-1NaCl是进行棉花耐盐性鉴定的适宜盐浓度.棉花的耐盐性在生育期和品种间表现不同:中棉所44和中棉所177是在萌发期和苗期均表现稳定的耐盐品种,表现稳定但不耐盐品种有中棉所102、苏棉12号和泗棉3号,表现稳定且中等耐盐品种有中棉所103、德夏棉1号和美棉33B;发芽率、发芽势、发芽指数、活力指数和鲜质量的盐害系数可以作为棉花萌发期耐盐鉴定指标,株高、叶片伸展速率、地上部干质量、根系干质量、根系活力和净光合速率的盐害系数可以作为棉花苗期耐盐鉴定指标.     

High‐efficient and precise base editing of C•G to T•A in the allotetraploid cotton (Gossypium hirsutum) genome using a modified CRISPR/Cas9 system

The base‐editing technique using CRISPR/nCas9 (Cas9 nickase) or dCas9 (deactivated Cas9) fused with cytidine deaminase is a powerful tool to create point mutations. In this study, a novel G. hirsutum‐Base Editor 3 (GhBE3) base‐editing system has been developed to create single‐base mutations in the allotetraploid genome of cotton (Gossypium hirsutum). A cytidine deaminase sequence (APOBEC) fused with nCas9 and uracil glycosylase inhibitor (UGI) was inserted into our CRISPR/Cas9 plasmid (pRGEB32‐GhU6.7). Three target sites were chosen for two target genes, GhCLA and GhPEBP, to test the efficiency and accuracy of GhBE3. The editing efficiency ranged from 26.67 to 57.78% at the three target sites. Targeted deep sequencing revealed that the C→T substitution efficiency within an ‘editing window’, approximately six‐nucleotide windows of ?17 to ?12 bp from the PAM sequence, was up to 18.63% of the total sequences. The 27 most likely off‐target sites predicted by CRISPR‐P and Cas‐OFFinder tools were analysed by targeted deep sequencing, and it was found that rare C→T substitutions (average < 0.1%) were detected in the editing windows of these sites. Furthermore, whole‐genome sequencing analyses on two GhCLA‐edited and one wild‐type plants with about 100× depth showed that no bona fide off‐target mutations were detectable from 1500 predicted potential off‐target sites across the genome. In addition, the edited bases were inherited to T1 progeny. These results demonstrate that GhBE3 has high specificity and accuracy for the generation of targeted point mutations in allotetraploid cotton.     

Cotton (Gossypium hirsutum) 14‐3‐3 proteins participate in regulation of fibre initiation and elongation by modulating brassinosteroid signalling

Cotton (Gossypium hirsutum) fibre is an important natural raw material for textile industry in the world. Understanding the molecular mechanism of fibre development is important for the development of future cotton varieties with superior fibre quality. In this study, overexpression of Gh14‐3‐3L in cotton promoted fibre elongation, leading to an increase in mature fibre length. In contrast, suppression of expression of Gh14‐3‐3L, Gh14‐3‐3e and Gh14‐3‐3h in cotton slowed down fibre initiation and elongation. As a result, the mature fibres of the Gh14‐3‐3 RNAi transgenic plants were significantly shorter than those of wild type. This ‘short fibre’ phenotype of the 14‐3‐3 RNAi cotton could be partially rescued by application of 2,4‐epibrassinolide (BL). Expression levels of the BR‐related and fibre‐related genes were altered in the Gh14‐3‐3 transgenic fibres. Furthermore, we identified Gh14‐3‐3 interacting proteins (including GhBZR1) in cotton. Site mutation assay revealed that Ser163 in GhBZR1 and Lys51/56/53 in Gh14‐3‐3L/e/h were required for Gh14‐3‐3‐GhBZR1 interaction. Nuclear localization of GhBZR1 protein was induced by BR, and phosphorylation of GhBZR1 by GhBIN2 kinase was helpful for its binding to Gh14‐3‐3 proteins. Additionally, 14‐3‐3‐regulated GhBZR1 protein may directly bind to GhXTH1 and GhEXP promoters to regulate gene expression for responding rapid fibre elongation. These results suggested that Gh14‐3‐3 proteins may be involved in regulating fibre initiation and elongation through their interacting with GhBZR1 to modulate BR signalling. Thus, our study provides the candidate intrinsic genes for improving fibre yield and quality by genetic manipulation.     

Genome-wide association study for candidate genes controlling seed yield and its components in rapeseed (Brassica napus subsp. napus)

Genetic improvement of seed yield per plant (SY) is one of the major objectives in Brassica napus breeding programme. SY, being a complex quantitative trait is directly and indirectly influenced by yield-component traits such as siliqua length (SL), number of seeds per siliqua (NSS), and thousand seed weight (TSW). Therefore, concurrent improvement in SL, NSS and TSW can lead to higher SY in B. napus. This study was conducted to identify significant SNPs and putative candidate genes governing SY and its component traits (SL, NSS, TSW). All these traits were evaluated in a diverse set of 200 genotypes representing diversity from wide geographical locations. Of these, a set of 125 genotypes were chosen based on pedigree diversity and multi-location trait variation for genotyping by sequencing (GBS). Best linear unbiased predictors (BLUPs) of all the traits were used for genome-wide association study (GWAS) with 85,126 SNPs obtained from GBS. A total of 16, 18, 27 and 18 SNPs were found to be significantly associated for SL, NSS, TSW and SY respectively. Based on linkage disequilibrium decay analysis, 150 kb genomic region flanking the SNP was used for the identification of underlying candidate genes for each test trait. Important candidate genes involved in phytohormone signaling (WAT1, OSR1, ARR8, CKX1, REM7, REM9, BG1) and seed storage proteins (Cruciferin) were found to have significant influence on seed weight and yield. Genes involved in sexual reproduction and fertilization (PERK7, PERK13, PRK3, GATA15, NFD6) were found to determine the number of seeds per siliqua. Several genes found in this study namely ATS3A, CKX1, SPL2, SPL6, SPL9, WAT1 showed pleiotropic effect with yield component traits. Significant SNPs and putative candidate genes identified for SL, NSS, TSW and SY could be used in marker-assisted breeding for improvement of crop yield in B. napus. Genotypes identified with high SL, NSS, TSW and SY could serve as donors in crop improvement programs in B. napus.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-01060-9.     

Managing pluvial resources and improving soil hydrology: Survival questions for cotton farming systems. The case study of the Mouhoun (Burkina Faso)

Cotton cropping has been developing for more than 40 years in the western part of Burkina Faso. It has made a definite modernization of the traditional farming system. Modernization is illustrated by adoption of specific agricultural practices like monoculture, tillage, straight sowing and slope ridge planting. Misuse and non-adaptable local pedologic and climatic context of these new practices perturb soil hydrological processes. Outcomes are water loss by runoff, erosion and changes in the soils physical and physiochemical properties.Besides, we can notice in this area for more than 30 years climatic changes which require us to ask ourselves what are the resulting consequences. In fact, these climatic changes generate an extension of the rainy season, an increase in the frequency of high intensity spells but also recurrence of dry spells during the rainy season. Do these new climatic conditions exacerbate the consequences of cotton practices on hydrological processes and induce an aggravation of flow and erosion processes?These evolutions have direct consequences on crop production whereas needs are highly increasing. The improvement of the prevailing agricultural practices and innovative practices might provide improved pluvial resources in critical moments such as rainfall excess at the beginning and the end of the rainy season and short drought conditions after sowing or germination and during flowering. However two questions arise. In the rainfed agriculture, which cultural practices (to reduce runoff and to favour infiltration) are, in regard to pedologic, climatic or socio-economic context, able to allow improved water efficiency and, as a result, an increase of the food grains production? Might selected practices and production addition satisfy requirements of the coming population?     

Application of a probabilistic model for analysing the abortion of seeds and pods in winter oilseed rape (Brassica napus)

In winter oilseed rape (WOSR), only a subset of ovules can develop into seeds in the majority of pods. Any difficulty during the process of seed production may result in seed or pod abortion. This study aimed to reproduce the process of seed development in WOSR based on a limited number of parameters. As a result of the complexity of the developmental patterns of WOSR, it is challenging to identify the roles of various factors that influence seed production using an experimental approach. Here, we present a stochastic probabilistic model of seed development. The generalised least squares method was implemented to estimate the model parameters using the experimental data. Experiments were done in Grignon (France) in 2008 and 2009. The variations in the parameters were analysed according to the following four factors: year, pod rank, inflorescence position and ramification‐clipping treatment. The year had no effect on the number of ovules per ovary (μ) and the probability of seed viability (p). The proportion of effective pollen grains (k) significantly decreased with pod rank at the end of the main stem. Inflorescence position influenced the number of ovules per ovary (μ: 30.8–33.8 from top to bottom) and the parameter k. The mean number of seeds per pod on the main stem and the bottom ramification along the stem was larger than the other ramifications within one plant. Ramification‐clipping treatment increased the number of ovules per ovary (μ: 31 for control plants and 32 for clipped plants), the parameter k and the number of seeds per pod (p). This effect could be due to the competition for assimilates between the pods and seeds. Furthermore, the distribution parameters of the pollen number per stigma (m) remained stable, and the probability of pod survival (Bo) varied with different factors, including the year, pod rank and inflorescence position. Our results indicate that pollen germination is a factor that determines final seed number. This model can identify the impact of each of the factors that lead to the abortion of seeds and pods in WOSR, which include the position effect, assimilate competition and pollination limitation. However, further studies on the pollination process in WOSR should help to refine this model.     

苗期紫花苜蓿株体对不同地区垂穗披碱草种子萌发生长的化感作用

以发芽率、苗长、根长、苗干重、根干重变化为种子萌发和幼苗生长参数,研究苗期紫花苜蓿(Medicago sativa)植株浸提液对不同地区垂穗披碱草(Elymus nutans)种子萌发生长的化感作用。结果表明:地上部浸提液对LS、DX垂穗披碱草种子发芽率具有明显的促进作用,而对GS、KMX、LKZ、LZ地区垂穗披碱草种子发芽率均表现为抑制作用,其中对GS垂穗披碱草种子发芽率的抑制作用最强,在14.5%和5.5%浓度处理时抑制率分别为57.89%、55.26%;苗长方面,浸提液5.5%浓度对垂穗披碱草苗长的抑制率顺序为:LZNQDXLSKMXQHGSLKZ,14.5%处理时抑制率顺序为:LZKMXLKZNQGSQHDXLS,其中抑制率最高的为LZ垂穗披碱草,在14.5%和5.5%浓度处理时抑制率分别为33.03%、28.97%;根长方面,5.5%处理对垂穗披碱草根长的抑制率顺序为:QHNQLZKMX、GSLKZDX、LS,14.5%处理时抑制率顺序为:GSQH、NQLZLSKMXDXLKZ,其中在高浓度下抑制率最高的为GS垂穗披碱草,抑制率为57.69%;地上部浸提液对LKZ、LZ垂穗披碱草苗干重均具有促进作用,高浓度浸提液对NQ垂穗披碱草苗干重产生促进作用(RI0),而对KMX、DX垂穗披碱草苗干重均表现为抑制作用;根干重方面,浸提液对LS、QH、GS、NQ、LKZ垂穗披碱草根干重均有明显的抑制作用,而对KMX、DX垂穗披碱草根干重产生促进作用,高浓度浸提液对LZ垂穗披碱草的根干重产生促进作用。从根浸提液的作用来看,根浸提液除对LS、DX垂穗披碱草种子发芽率和根干重、GS垂穗披碱草种子发芽率和苗干重及NQ垂穗披碱草根干重具有促进作用外(P 0.05),对其余地区垂穗披碱草的各项指标均有明显的抑制作用(RI0)。所有以上结果表明,紫花苜蓿植株浸提液对垂穗披碱草种子萌发生长的作用具有一定的浓度效应。不同地区垂穗披碱草对紫花苜蓿地上部浸提液的敏感性趋势总体为:GSQHLSKMXNQLKZLZ,最不敏感或有促进作用的是DX垂穗披碱草;对根浸提液的敏感性趋势总体为:QHNQLZKMX,根浸提液对LS、GS、LKZ、DX垂穗披碱草种子萌发生长具有促进作用。紫花苜蓿植株不同部位浸提液对垂穗披碱草种子萌发生长的化感效应顺序为:地上部根。     

Raman study of poly(alanine-glycine)-based peptides containing tyrosine, valine, and serine as model for the semicrystalline domains of Bombyx mori silk fibroin

For a deeper insight into the structure of Bombyx mori silk fibroin, some model peptides containing tyrosine (Y), valine (V), and serine (S) in the basic (AG)n sequence were synthesized by the solid-phase method and analyzed by Raman spectroscopy in order to clarify their conformation and to evaluate the formation and/or disruption of the ordered structure typical of B. mori silk fibroin upon incorporation of Y, V, and S residues into the basic (AG)n sequence. The Raman results indicated that the silk I structure remains stable only when the Y residue is positioned near the chain terminus; otherwise, a silk I --> silk II conformational transition occurs. The peptides AGVGAGYGAGVGAGYGAGVGAGYG(AG)3 and (AG)3YG(AG)2VGYG(AG)3YG(AG)3 treated with LiBr revealed a prevalent silk II conformation; moreover, the former contained a higher amount of random coil than the latter. This result was explained in relation to the different degrees of interruption of the (AG)n sequence. The Raman analysis of the AGSGAG-containing samples confirmed that the AGSGAG hexapeptide is a good model for the silk II crystalline domain. As the number of AGSGAG repeating units decreased, the random coil content increased. The study of the Y domain (I850/I830 intensity ratio) allowed us to hypothesize that in the packing characteristic of Silk I and Silk II conformations the Y residues experience different environments and hydrogen-bonding arrangements; the packing typical of silk I structure traps the tyrosyl side chains in environments more unfavorable to phenoxyl hydrogen-bonding interactions.     

苜蓿根瘤菌17560细胞膜膜脂组成分析及DGTS合成基因克隆与表达

【目的】分析一株分离自黑龙江省的苜蓿根瘤菌在低磷胁迫及正常磷含量条件下细胞膜脂的组成,并从该菌中克隆和鉴定细胞膜无磷脂二酰基甘油三甲基高丝氨酸(DGTS)合成基因。【方法】分别在不同磷含量的Sherwood基本培养基中进行根瘤菌培养,采用Bligh-Dyer方法提取细胞膜脂,以文献报道Sinorhizobium meliloti(苜蓿中华根瘤菌)菌株1021的脂类图谱和磷脂PE、PG、PC标准品作为参照,利用薄层层析方法分析不同磷含量条件下培养菌株的细胞膜脂组成。根据GenBank中已发表的DGTS合成基因btaA和btaB序列设计引物,以产DGTS菌株基因组DNA为模板,扩增btaA和btaB同源基因,并在E.coil BL21(DE3)表达。同时检测表达菌株是否合成细胞膜无磷脂DGTS以验证基因功能。对菌株17560进行16S rRNA基因序列分析。【结果】分离自黑龙江省的苜蓿根瘤菌17560与Sinorhizobium meliloti的16S rRNA基因序列相似性高达99.8%,但其细胞膜脂组成明显不同于参比菌株Sinorhizobium meliloti 1021的膜脂组成。在低磷胁迫条件下,该菌株的细胞膜脂主要由OL和DGTS等无磷脂组成,但OL的组成明显不同,该菌株含有3种不同类型的鸟氨酸脂(OLs),而参比菌株Sinorhizobium meliloti 1021只含有一种类型的鸟氨酸脂(OL)。在正常磷含量条件下,该菌株的细胞膜脂主要由PE和一种未知的含氨基磷脂组成,PG与PC的含量均较少,而参比菌株Sinorhizobium meliloti 1021的细胞膜脂主要由PE、PG与PC组成。通过PCR扩增从产DGTS菌株17560中获得1 913 bpDNA片段,经序列分析发现其中有两个ORF与菌株Sinorhizobium meliloti 1021的btaA和btaB基因序列相似性均为99%。将该DNA片段克隆于pET-30a(+)得到重组质粒pLH01,转化宿主菌获得表达菌株E.coli BL21(DE3).pLH01,经IPTG诱导后产生相对分子量约为45 kD和25 kD的蛋白。薄层层析验证重组菌细胞膜脂组成,结果表明,表达菌株E.coliBL21(DE3).pLH01可以在IPTG诱导后合成无磷脂DGTS,而转入空载体pET-30a(+)的阴性对照菌株E.coli BL21(DE3).pET-30a(+)则不能合成。【结论】系统发育地位相同的苜蓿根瘤菌株的细胞膜脂组成明显不同;苜蓿根瘤菌的细胞膜组成随培养基中的磷含量不同而变化,低磷胁迫条件下其细胞膜脂主要由OL和DGTS等无磷脂组成;在Sinorhizobium膜脂中首次发现一种未知的氨基磷脂及3种不同类型的鸟氨酸脂(OLs);从菌株17560中克隆获得2个DGTS合成基因btaA和btaB,在大肠杆菌中成功表达,并证实了所表达基因的功能。