Small RNA and degradome deep sequencing reveals important roles of microRNAs in cotton (Gossypium hirsutum L.) response to root-knot nematode Meloidogyne incognita infection.

Investigation of cotton response to nematode infection will allow us to better understand the cotton immune defense mechanism and design a better biotechnological approach for efficiently managing pest nematodes in cotton. In this study, we firstly treated cotton by root knot nematode (RKN, Meloidogyne incognita) infections, then we employed the high throughput deep sequencing technology to sequence and genome-widely identify all miRNAs in cotton; finally, we analyzed the functions of these miRNAs in cotton response to RKN infections. A total of 266 miRNAs, including 193 known and 73 novel miRNAs, were identified by deep sequencing technology, which belong to 67 conserved and 66 novel miRNA families, respectively. A majority of identified miRNA families only contain one miRNA; however, miR482 family contains 14 members and some others contain 2-13 members. Certain miRNAs were specifically expressed in RKN-infected cotton roots and others were completely inhibited by RKN infection. A total of 50 miRNAs were differentially expressed after RKN infection, in which 28 miRNAs were up-regulated and 22 were inhibited by RKN treatment. Based on degradome sequencing, 87 gene targets were identified to be targeted by 57 miRNAs. These miRNA-targeted genes are involved in the interaction of cotton plants and nematode infection. Based on GO (gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis, 466 genes from all 636 miRNA targets were mapped to 6340 GO terms, 181 genes from 228 targets of differentially expressed miRNAs were mapped to 1588 GO terms. The GO terms were then categorized into the three main GO classes: biological processes, cellular components, and molecular functions. The targets of differentially expressed miRNAs were enriched in 43 GO terms, including 22 biological processes, 10 cellular components, and 11 molecular functions (p < 0.05). Many identified processes were associated with organism responses to the environmental stresses, including regulation of nematode larval development, response to nematode, and response to flooding. Our results will enhance the study and application of developing new cotton cultivars for nematode resistance.

Functional PPO2 mutations: co-occurrence in one plant or the same ppo2 allele of herbicide-resistant Amaranthus palmeri in the US mid-south.

BACKGROUND: Protoporphyrinogen IX oxidase 2 (PPO2) inhibitors are important for the management of glyphosate- and acetolactate synthase-resistant Palmer amaranth [Amaranthus palmeri (S.) Wats.]. The evolving resistance to PPO inhibitors is of great concern. We surveyed the evolution of resistance to fomesafen in the US Mid-south and determined its correlation with the known functional PPO2 target-site mutations (TSM). RESULTS: The 167 accessions analyzed were grouped into five categories, four resistant (147) and one susceptible (20). Arkansas accessions constituted 100% of the susceptible group while the Missouri accessions comprised 60% of the most resistant category. The majority of Mississippi accessions (88%) clustered in the high-survival-high-injury category, manifesting an early-stage resistance evolution. One hundred and fifteen accessions were genotyped for four known TSMs; 74% of accessions carried at least one TSM. The most common single TSM was ΔG210 (18% of accessions) and the predominant double mutation was ΔG210 + G399A (17%). Other mutations are likely less favorable, hence are rare. All TSMs were detected in three accessions. Further examination revealed that 9 and two individuals carried G399A + G210 and G399A + R128G TSM in the same allele, respectively. The existence of these combinations is supported by molecular modeling. CONCLUSIONS: Resistance to PPO inhibitors is widespread across the Mid-southern USA. Highly resistant field populations have plants with multiple mutations. G399A is the most prone to co-occur with other ppo2 mutations in the same allele. Mutation at R128 in the configuration of the PPO2 catalytic domain restrains the co-occurrence of R128G with ΔG210, making ΔG210 + G399A the most plausible, tolerable functional mutation combination to co-occur in the same ppo2 allele.

Characterization of Current Fusarium oxysporum f. sp. vasinfectum Isolates from Cotton in the San Joaquin Valley of California and Lower Valley El Paso, Texas.

Fusarium oxysporum f. sp. vasinfectum race 4 is a causal agent of Fusarium wilt of cotton (Gossypium spp.). This study aimed to characterize the existing distribution and frequency of current field populations of F. oxysporum f. sp. vasinfectum race 4 genotypes in the San Joaquin Valley (SJV) of California and Lower Valley El Paso, TX and examine representative isolates for aggressiveness during different stages of seedling development. A survey was conducted from 2017 to 2019 across 13 locations in the SJV and one location in El Paso, TX during 2018. From the SJV, isolates identified as the F. oxysporum f. sp. vasinfectum race 4 T genotype were dispersed across the SJV, whereas isolates identified as the F. oxysporum f. sp. vasinfectum race 4 N genotype were most frequently isolated from cotton fields in the northern county of Merced. The F. oxysporum f. sp. vasinfectum race 4 isolates from the Texas location were identified as the MT genotype. A selection of representative isolates was evaluated using three inoculation assays (rolled-towel, F. oxysporum f. sp. vasinfectum-infested oat seed, and root-dip inoculation) to test the isolates' abilities to produce symptoms during seedling stages of cotton development. All isolates tested were capable of producing symptoms on cotton; however, isolate aggressiveness varied within and across inoculation assays. In all assays, higher levels of disease development were observed in the moderately susceptible Pima (Gossypium barbadense L.) cultivars (DP-340 or PHY-830) when compared with the moderately tolerant Upland (G. hirsutum L.) cultivar (FM-2334). However, no correlation was found among the different response variables for the rolled-towel assay when compared with the root-dip and infested oat seed assays. These results suggest that different genes are involved in the resistance response during the early seedling development stage measured in the rolled-towel assay compared with the later seedling development stages measured during the root-dip inoculation and infested oat seed assays, revealing the complexity of the Fusarium wilt disease and host-plant resistance mechanisms.

Sequence Composition of Bacterial Chromosome Clones in a Transgressive Root-Knot Nematode Resistance Chromosome Region in Tetraploid Cotton.

Plants evolve innate immunity including resistance genes to defend against pest and pathogen attack. Our previous studies in cotton (Gossypium spp.) revealed that one telomeric segment on chromosome (Chr) 11 in G. hirsutum cv. Acala NemX (rkn1 locus) contributed to transgressive resistance to the plant parasitic nematode Meloidogyne incognita, but the highly homologous segment on homoeologous Chr 21 had no resistance contribution. To better understand the resistance mechanism, a bacterial chromosome (BAC) library of Acala N901 (Acala NemX resistance source) was used to select, sequence, and analyze BAC clones associated with SSR markers in the complex rkn1 resistance region. Sequence alignment with the susceptible G. hirsutum cv. TM-1 genome indicated that 23 BACs mapped to TM-1-Chr11 and 18 BACs mapped to TM-1-Chr 21. Genetic and physical mapping confirmed less BAC sequence (53-84%) mapped with the TM-1 genome in the rkn1 region on Chr 11 than to the homologous region (>89%) on Chr 21. A 3.1-cM genetic distance between the rkn1 flanking markers CIR316 and CIR069 was mapped in a Pima S-7 × Acala NemX RIL population with a physical distance ∼1 Mbp in TM-1. NCBI Blast and Gene annotation indicated that both Chr 11 and Chr 21 harbor resistance gene-rich cluster regions, but more multiple homologous copies of Resistance (R) proteins and of adjacent transposable elements (TE) are present within Chr 11 than within Chr 21. (CC)-NB-LRR type R proteins were found in the rkn1 region close to CIR316, and (TIR)-NB-LRR type R proteins were identified in another resistance rich region 10 cM from CIR 316 (∼3.1 Mbp in the TM-1 genome). The identified unique insertion/deletion in NB-ARC domain, different copies of LRR domain, multiple copies or duplication of R proteins, adjacent protein kinases, or TE in the rkn1 region on Chr 11 might be major factors contributing to complex recombination and transgressive resistance.

Characterization of the Complete Genome and P0 Protein for a Previously Unreported Genotype of Cotton Leafroll Dwarf Virus, an Introduced Polerovirus in the United States.

Virus-like disease symptoms consisting of leaf cupping, shortened internodes, and overall stunting were observed in commercial cotton fields in Alabama in 2017 to 2018. To determine the complete genome sequence of the suspected causal polerovirus, symptomatic leaf samples were collected in Macon County, Alabama, and subjected to Illumina RNA sequencing. Based on BLASTn analysis, the Illumina contig of 5,771 nt shared the highest nucleotide identity (approximately 95%) with members of the species Cotton leafroll dwarf virus (CLRDV) (genus Polerovirus; family Luteoviridae) from Argentina and Brazil. The full-length viral genome sequence was verified by reverse transcription (RT)-PCR amplification, cloning, and Sanger sequencing. The complete CLRDV genome of 5,865 nt in length shared 94.8 to 95.2% nucleotide identity with six previously reported CLRDV isolates. The genome of the CLRDV isolate amplified from Alabama samples (CLRDV-AL) has seven predicted open reading frames (ORFs). Viral proteins 1 to 5 (P1 to P5) shared 91.9 to 99.5% amino acid identity with the six CLRDV isolates from Argentina and Brazil. However, P0, the suppressor of host gene silencing, shared 82.4 to 88.5% pairwise amino acid identity with the latter CLRDV isolates. Phylogenetic analysis of the seven full-length CLRDV genomes resolved three sister clades: CLRDV-AL, CLRDV-typical, and CLRDV-atypical, respectively. Three recombination events were detected by the recombination detection program among the seven CLRDV isolates with breakpoints occurring along the genome. Pairwise nucleotide identity comparisons of ORF0 sequences for the three CLRDV-AL field isolates indicated that they were >99% identical, suggesting that this previously unknown CLRDV genotype represents a single introduction to Alabama.

The effect of two QTLs for resistance to Meloidogyne incognita in cotton on nematode egression from roots.

Cotton is widely grown in the southern US and Meloidogyne incognita is its most significant pathogen. The germplasm line M-120 RNR is highly resistant to M. incognita due to two resistance QTLs (quantitative trait loci), qMi-C11 and qMi-C14. Both QTLs reduce total egg production, but the QTLs affect M. incognita development at different life stages. The QTLs do not appear to affect initial penetration of M. incognita but genotypes containing qMi-C11 had fewer nematodes in the roots 8 days after inoculation than near isolines without qMi-C11, which may indicate M. incognita egression from roots. Three greenhouse trials were conducted using cotton isolines to determine whether qMi-C11 and qMi-C14 affect egression of M. incognita juveniles from roots. On each of the five sampling dates (4, 6, 8, 10, and 12 DAI), nematodes that egressed from roots were counted and roots were stained to count nematodes that remained in the roots. The effect of resistance QTLs on M. incognita egression from the roots differed among the trials. Nematode egression was consistently numerically greater, but inconsistently statistically different, from plants with both QTLs than from plants with neither QTL. Plants with only one QTL generally did not differ from plants with both QTLs, and the effects of qMi-C11 and qMi-C14 did not differ in any consistent way. In a separate experiment, plants with neither QTL had more eggs per egg mass than did plants with both QTLs, whereas plants with only one QTL had an intermediate number. Root gall size was measured in two trials and no consistent differences in gall size were observed. We conclude that (1) qMi-C11 and qMi-C14 do not stimulate nematode egression from cotton roots, (2) both qMi-C11 and qMi-C14 reduce M. incognita eggs/egg mass, and (3) neither qMi-C11 nor qMi-C14 affect gall size.

Small RNA Sequencing Reveals Regulatory Roles of MicroRNAs in the Development of Meloidogyne incognita.

MicroRNAs (miRNAs) are an extensive class of small regulatory RNAs. Knowing the specific expression and functions of miRNAs during root-knot nematode (RKN) (Meloidogyne incognita) development could provide fundamental information about RKN development as well as a means to design new strategies to control RKN infection, a major problem of many important crops. Employing high throughput deep sequencing, we identified a total of 45 conserved and novel miRNAs from two developmental stages of RKN, eggs and J2 juveniles, during their infection of cotton (Gossypium hirsutum L.). Twenty-one of the miRNAs were differentially expressed between the two stages. Compared with their expression in eggs, two miRNAs were upregulated (miR252 and miRN19), whereas 19 miRNAs were downregulated in J2 juveniles. Nine miRNAs were expressed at high levels, with >1000 reads per mapped million (RPM) sequenced reads in both eggs and J2 juveniles (miR1, miR124, miR2-3p, miR252, miR279, miR57-5p, miR7904, miR87, and miR92). Three miRNAs were only expressed in eggs (miR4738, miRN3, and miRN5). These differentially expressed miRNAs may control RKN development by regulating specific protein-coding genes in pathways associated with RKN growth and development.

Transcriptome analysis of a nematode resistant and susceptible upland cotton line at two critical stages of Meloidogyne incognita infection and development.

Host plant resistance is the most practical approach to control the Southern root-knot nematode (Meloidogyne incognita; RKN), which has emerged as one of the most serious economic pests of Upland cotton (Gossypium hirsutum L.). Previous QTL analyses have identified a resistance locus on chromosome 11 (qMi-C11) affecting galling and another locus on chromosome-14 (qMi-C14) affecting egg production. Although these two QTL regions were fine mapped and candidate genes identified, expression profiling of genes would assist in further narrowing the list of candidate genes in the QTL regions. We applied the comparative transcriptomic approach to compare expression profiles of genes between RKN susceptible and resistance genotypes at an early stage of RKN development that coincides with the establishment of a feeding site and at the late stage of RKN development that coincides with RKN egg production. Sequencing of cDNA libraries produced over 315 million reads of which 240 million reads (76%) were mapped on to the Gossypium hirsutum genome. A total of 3,789 differentially expressed genes (DEGs) were identified which were further grouped into four clusters based on their expression profiles. A large number of DEGs were found to be down regulated in the susceptible genotype at the late stage of RKN development whereas several genes were up regulated in the resistant genotype. Key enriched categories included transcription factor activity, defense response, response to phyto-hormones, cell wall organization, and protein serine/threonine kinase activity. Our results also show that the DEGs in the resistant genotype at qMi-C11 and qMi-C14 loci displayed higher expression of defense response, detoxification and callose deposition genes, than the DEGs in the susceptible genotype.

Detection and Characterization of Fusarium oxysporum f. sp. vasinfectum VCG0114 (Race 4) Isolates of Diverse Geographic Origins.

A highly virulent cotton wilt pathogen, Fusarium oxysporum f. sp. vasinfectum VCG0114 (race 4) was found in West Texas in 2017, after being known in California since 2001. Isolates obtained from wilted plants collected in 2017 from Texas, in 2015 from China, and during 2001 to 2014 from California and isolates from historical collections including the race 4 reference isolate were characterized by soil-infestation pathogenicity assays, DNA sequence analysis, and vegetative compatibility analysis. All obtained F. oxysporum f. sp. vasinfectum isolates belonged to VCG0114. All of these isolates, except one isolate from China, caused disease in a soil-infestation assay without nematodes. Thus, they belong to the nematode-independent pathotype. Texas isolates were significantly more virulent than were isolates from China or California on Gossypium barbadense 'Pima S-7'. Four different genotypes (N, T, MT, and MiT) were identified based on the transposable element Tfo1 insertion into the PHO gene and independent MULE or MITE insertions into the Tfo1 transposon. Some significant differences in virulence were detected among the genotypes in some locations. No differences in pathogenicity were observed between the California and China collection isolates on Pima S-7, and the virulence of the major genotypes was similar on the Gossypium hirsutum cultivar 'Stoneville 474' or the Barbren 713 germplasm line. Simple polymerase chain reaction (PCR) methods were developed to specifically determine and detect the four genotypes within VCG0114. A specific PCR method to detect all VCG0114 isolates was also developed. These methods will facilitate the timely identification of infested fields and seed lots and the elucidation of evolutionary relationships among the isolates. This should help to closely monitor the movement of the pathogen and reduce dissemination of these devastating pathogens.

Fusarium wilt of cotton may commonly result from the interaction of Fusarium oxysporum f. sp. vasinfectum with Belonolaimus longicaudatus.

The interaction between Fusarium oxysporum f. sp. vasinfectum (Fov) and Meloidogyne incognita (root-knot nematode) resulting in Fusarium wilt (FW) of cotton is well-known. Although Belonolaimus longicaudatus (sting nematode) can also interact with Fov and cause FW, it has long been believed that virtually all of the FW in Georgia is caused by the interaction of Fov with M. incognita. In recent years, FW has been reported more frequently in Georgia, which suggests that something affecting the disease complex may have changed. In 2015 and 2016, a survey of 27 Georgia cotton fields in 10 counties was conducted. At least 10 soil and stem samples per field were collected from individual plants showing symptoms of FW to quantify plant-parasitic nematode levels and identify Fov races. Fov race 1 was identified in all samples in 2015, but one sample also had the LA110 genotype and another sample also had the LA108 genotype. In 2016, all Fov races and genotypes found in 2015 were present, however, MDS-12 and LA127/140 also were found. Meloidogyne incognita was present in 18% of fields in 2015 and 40% in 2016, whereas B. longicaudatus was present in all fields in 2015 and 75% of fields in 2016. Regardless of whether they occurred separately or together, M. incognita and B. longicaudatus were present, respectively, in 18% and 55% of individual samples in 2015 and 40% and 51% in 2016. However, M. incognita without B. longicaudatus was found in 7% of samples in 2015 and 34% in 2016, whereas B. longicaudatus without M. incognita was found in 45% of samples in 2015 and 44% in 2016. We conclude that Fov race 1 continues to be the dominant race in Georgia and many instances of FW in Georgia may be due to Fov interacting with B. longicaudatus and not M. incognita as previously believed.The interaction between Fusarium oxysporum f. sp. vasinfectum (Fov) and Meloidogyne incognita (root-knot nematode) resulting in Fusarium wilt (FW) of cotton is well-known. Although Belonolaimus longicaudatus (sting nematode) can also interact with Fov and cause FW, it has long been believed that virtually all of the FW in Georgia is caused by the interaction of Fov with M. incognita. In recent years, FW has been reported more frequently in Georgia, which suggests that something affecting the disease complex may have changed. In 2015 and 2016, a survey of 27 Georgia cotton fields in 10 counties was conducted. At least 10 soil and stem samples per field were collected from individual plants showing symptoms of FW to quantify plant-parasitic nematode levels and identify Fov races. Fov race 1 was identified in all samples in 2015, but one sample also had the LA110 genotype and another sample also had the LA108 genotype. In 2016, all Fov races and genotypes found in 2015 were present, however, MDS­12 and LA127/140 also were found. Meloidogyne incognita was present in 18% of fields in 2015 and 40% in 2016, whereas B. longicaudatus was present in all fields in 2015 and 75% of fields in 2016. Regardless of whether they occurred separately or together, M. incognita and B. longicaudatus were present, respectively, in 18% and 55% of individual samples in 2015 and 40% and 51% in 2016. However, M. incognita without B. longicaudatus was found in 7% of samples in 2015 and 34% in 2016, whereas B. longicaudatus without M. incognita was found in 45% of samples in 2015 and 44% in 2016. We conclude that Fov race 1 continues to be the dominant race in Georgia and many instances of FW in Georgia may be due to Fov interacting with B. longicaudatus and not M. incognita as previously believed.

Resistance Quantitative Trait Loci qMi-C11 and qMi-C14 in Cotton Have Different Effects on the Development of Meloidogyne incognita, the Southern Root-Knot Nematode.

Quantitative trait loci (QTLs) qMi-C11 and qMi-C14 impart a high level of resistance to Meloidogyne incognita in cotton. Breeders had previously backcrossed both QTLs into the susceptible Coker 201 to create the highly resistant M-120 RNR, and we crossed Coker 201 and M-120 RNR to create near-isogenic lines with either qMi-C11 or qMi-C14. Previous work suggests different modes of action for qMi-C11 and qMi-C14. To document individual and combined effects of the QTLs on nematode development and reproduction, Coker 201 (neither QTL), M-120 RNR (both QTLs), CH11 near isoline (qMi-C11), and CH14 near isoline (qMi-C14) were inoculated with M. incognita. At 4, 8, 12, 16, 20, 25, and 30 days after inoculation (DAI), roots were stained to observe nematode developmental stages (second-stage juvenile [J2], swollen second-stage juvenile [SJ2], third-stage juvenile [J3], fourth-stage juvenile [J4], and female), and the number of galls was counted. At 20, 25, 30, and 40 DAI, M. incognita eggs were harvested and counted. At 30 DAI, 80% of the nematodes on Coker 201 were female compared with 50, 40, and 33% females on CH14, CH11, and M-120 RNR, respectively, and greater proportions of nematodes remained in J2 in M-120 RNR (41%), CH11 (58%), and CH14 (27%) than in Coker 201 (9%). More nematodes progressed to J3 or J4 on Coker 201 and CH14 than on CH11 or M-120 RNR. Coker 201 and CH14 had more galls than M-120 RNR. Coker 201 had more eggs than the other genotypes at 30 DAI. Nematode development beyond J2 or SJ2 was significantly reduced by qMi-C11, and development beyond J3 or J4 was significantly reduced by qMi-C14. This study confirms that qMi-C11 and qMi-C14 act at different times and have different effects on the development of M. incognita, and therefore, they have different modes of action.

Development of a qPCR Protocol to Detect the Cotton Bacterial Blight Pathogen, Xanthomonas citri pv. malvacearum, from Cotton Leaves and Seeds.

Bacterial blight, historically a seed-borne disease of cotton (Gossypium hirsutum) is caused by Xanthomonas citri pv. malvacearum, resulted in significant economic losses prior to development of resistant varieties and implementation of acid-delinting of planting seed. Periodic outbreaks have been associated with seed since the early twentieth century; of note, the disease has experienced a resurgence since 2011. Effective management of bacterial blight is dependent on accurate diagnosis and detection of the pathogen. Currently, detection of X. citri pv. malvacearum is performed by time-consuming microbiological methods. In this study, a novel and sensitive TaqMan-based qPCR protocol was developed to test for X. citri pv. malvacearum in cotton plant tissue. The primers developed are specific to five races of X. citri pv. malvacearum, but not to other Xanthomonas species or cotton-associated nonpathogenic bacteria. The efficiency of this assay was evaluated on artificially inoculated cotton leaves and seed, on naturally infected cotton leaves, and on bolls and seed originating from bacterial blight symptomatic bolls. The protocol's efficiency from artificially inoculated plant tissue was 102 copies g-1 and 37 copies from 1 g seed for leaves and seed, respectively. In addition, X. citri pv. malvacearum was detected from 94% of the seed samples originating from blight symptomatic bolls. The qPCR protocol provides a rapid and accurate method for diagnosis and detection of bacterial blight and offers a tool for monitoring X. citri pv. malvacearum and potentially reducing its spread in seed.

MicroRNA-target gene responses to root knot nematode (Meloidogyne incognita) infection in cotton (Gossypium hirsutum L.).

MicroRNAs (miRNAs) are a large class of small regulatory RNA molecules, however no study has been performed to elucidate the role of miRNAs in cotton (Gossypium hirsutum) response to the root knot nematode (RKN, Meloidogyne incognita) infection. We selected 28 miRNAs and 8 miRNA target genes to investigate the miRNA-target gene response to M. incognita infection. Our results show that RKN infection significantly affected the expression of several miRNAs and their targeted genes. After 10 days of RKN infection, expression fold changes on miRNA expressions ranged from down-regulated by 33% to upregulated by 406%; meanwhile the expression levels of miRNA target genes were 45.8% to 231%. Three miRNA-target pairs, miR159-MYB, miR319-TCP4 and miR167-ARF8, showed inverse expression patterns between gene targets and their corresponded miRNAs, suggesting miRNA-mediated gene regulation in cotton roots in response to RKN infection.

Differences in Active Defense Responses of Two Gossypium barbadense L. Cultivars Resistant to Fusarium oxysporum f. sp. vasinfectum Race 4.

A highly virulent race 4 genotype of Fusarium oxysporum f. sp. vasinfectum (Fov) was identified for the first time in the western hemisphere in 2002 in cotton fields in the San Joaquin Valley of California. The Gossypium barbadense L. cotton cultivars 'Seabrook Sea Island 12B2' ('SBSI') and 'Pima S-6' are resistant to Fov race 4. Active defense responses were quantitated by monitoring the accumulation of antimicrobial terpenoids (i.e., phytoalexins) in inoculated stem stele tissue in these cultivars. The increase in the concentration of the most toxic phytoalexins was statistically faster after 24 h in 'SBSI' compared to 'Pima S-6'. The sesquiterpenoid hemigossylic acid lactone, which was observed for the first time in nature, also accumulated in diseased plants. Neither hemigossylic acid lactone nor the disesquiterpenoids gossypol, gossypol-6-methyl ether, and gossypol-6,6'-dimethyl ether showed toxicity to Fov. Segregation of F2 progeny from 'SBSI' × 'Pima S-6' crosses gave a few highly susceptible plants and a few highly resistant plants, indicating separate genes for resistance in the two cultivars.

RNA-Seq transcriptome analysis of Amaranthus palmeri with differential tolerance to glufosinate herbicide.

Amaranthus palmeri (Amaranthaceae) is a noxious weed in several agroecosystems and in some cases seriously threatens the sustainability of crop production in North America. Glyphosate-resistant Amaranthus species are widespread, prompting the use of alternatives to glyphosate such as glufosinate, in conjunction with glufosinate-resistant crop cultivars, to help control glyphosate-resistant weeds. An experiment was conducted to analyze the transcriptome of A. palmeri plants that survived exposure to 0.55 kg ha-1 glufosinate. Since there was no record of glufosinate use at the collection site, survival of plants within the population are likely due to genetic expression that pre-dates selection; in the formal parlance of weed science this is described as natural tolerance. Leaf tissues from glufosinate-treated and non-treated seedlings were harvested 24 h after treatment (HAT) for RNA-Seq analysis. Global gene expression was measured using Illumina DNA sequence reads from non-treated and treated surviving (presumably tolerant, T) and susceptible (S) plants. The same plants were used to determine the mechanisms conferring differential tolerance to glufosinate. The S plants accumulated twice as much ammonia as did the T plants, 24 HAT. The relative copy number of the glufosinate target gene GS2 did not differ between T and S plants, with 1 to 3 GS2 copies in both biotypes. A reference cDNA transcriptome consisting of 72,780 contigs was assembled, with 65,282 sequences putatively annotated. Sequences of GS2 from the transcriptome assembly did not have polymorphisms unique to the tolerant plants. Five hundred sixty-seven genes were differentially expressed between treated T and S plants. Of the upregulated genes in treated T plants, 210 were more highly induced than were the upregulated genes in the treated S plants. Glufosinate-tolerant plants had greater induction of ABC transporter, glutathione S-transferase (GST), NAC transcription factor, nitronate monooxygenase (NMO), chitin elicitor receptor kinase (CERK1), heat shock protein 83, ethylene transcription factor, heat stress transcription factor, NADH-ubiquinone oxidoreductase, ABA 8'-hydroxylase, and cytochrome P450 genes (CYP72A, CYP94A1). Seven candidate genes were selected for validation using quantitative real time-PCR. While GST was upregulated in treated tolerant plants in at least one population, CYP72A219 was consistently highly expressed in all treated tolerant biotypes. These genes are candidates for contributing tolerance to glufosinate. Taken together, these results show that differential induction of stress-protection genes in a population can enable some individuals to survive herbicide application. Elevated expression of detoxification-related genes can get fixed in a population with sustained selection pressure, leading to evolution of resistance. Alternatively, sustained selection pressure could select for mutation(s) in the GS2 gene with the same consequence.

Microbial Resistance Mechanisms to the Antibiotic and Phytotoxin Fusaric Acid.

Fusaric acid (FA) produced by Fusarium oxysporum plays an important role in disease development in plants, including cotton. This non-specific toxin also has antibiotic effects on microorganisms. Thus, one expects a potential pool of diverse detoxification mechanisms of FA in nature. Bacteria and fungi from soils infested with Fusarium and from laboratory sources were evaluated for their ability to grow in the presence of FA and to alter the structure of FA into less toxic compounds. None of the bacterial strains were able to chemically modify FA. Highly FA-resistant strains were found only in Gram-negative bacteria, mainly in the genus of Pseudomonas. The FA resistance of the Gram-negative bacteria was positively correlated with the number of predicted genes for FA efflux pumps present in the genome. Phylogenetic analysis of predicted FA resistance proteins (FUSC, an inner membrane transporter component of the efflux pump) revealed that FUSC proteins having high sequence identities with the functionally characterized FA resistance protein FusC or Fdt might be the major contributors of FA resistance. In contrast, most fungi converted FA to less toxic compounds regardless of the level of FA resistance they exhibited. Five derivatives were detected, and the detoxification of FA involved either oxidative reactions on the butyl side chain or reductive reactions on the carboxylic acid group. The production of these metabolites from widely different phyla indicates that resistance to FA by altering its structure is highly conserved. A few FA resistant saprophytic or biocontrol strains of fungi were incapable of altering FA, indicating a possible involvement of efflux transporters. Deployment of both efflux and derivatization mechanisms may be a common feature of fungal FA resistance.

The genome of the cotton bacterial blight pathogen Xanthomonas citri pv. malvacearum strain MSCT1.

Xanthomonas citri pv. malvacearum is a major pathogen of cotton, Gossypium hirsutum L.. In this study we report the complete genome of the X. citri pv. malvacearum strain MSCT1 assembled from long read DNA sequencing technology. The MSCT1 genome is the first X. citri pv. malvacearum genome with complete coding regions for X. citri pv. malvacearum transcriptional activator-like effectors. In addition functional and structural annotations are presented in this study that will provide a foundation for future pathogenesis studies with MSCT1.

Detoxification of Fusaric Acid by the Soil Microbe Mucor rouxii.

Fusarium oxysporum f. sp. vasinfectum race 4 (VCG0114), which causes root rot and wilt of cotton (Gossypium hirsutum and G. barbadense), has been identified recently for the first time in the western hemisphere in certain fields in the San Joaquin Valley of California. This pathotype produces copious quantities of the plant toxin fusaric acid (5-butyl-2-pyridinecarboxylic acid) compared to other isolates of F. oxysporum f. sp. vasinfectum (Fov) that are indigenous to the United States. Fusaric acid is toxic to cotton plants and may help the pathogen compete with other microbes in the soil. We found that a laboratory strain of the fungus Mucor rouxii converts fusaric acid into a newly identified compound, 8-hydroxyfusaric acid. The latter compound is significantly less phytotoxic to cotton than the parent compound. On the basis of bioassays of hydroxylated analogues of fusaric acid, hydroxylation of the butyl side chain of fusaric acid may affect a general detoxification of fusaric acid. Genes that control this hydroxylation may be useful in developing biocontrol agents to manage Fov.

Genetic Diversity, Virulence, and Meloidogyne incognita Interactions of Fusarium oxysporum Isolates Causing Cotton Wilt in Georgia.

Locally severe outbreaks of Fusarium wilt of cotton (Gossypium spp.) in South Georgia raised concerns about the genotypes of the causal pathogen, Fusarium oxysporum f. sp. vasinfectum. Vegetative complementation tests and DNA sequence analysis were used to determine genetic diversity among 492 F. oxysporum f. sp. vasinfectum isolates obtained from 107 wilted plants collected from seven fields in five counties. Eight vegetative complementation groups (VCG) were found, with VCG 01117B and VCG 01121 occurring in 66% of the infected plants. The newly recognized VCG 01121 was the major VCG in Berrien County, the center of the outbreaks. All eight VCG resulted in significant increases in the percentages of wilted leaves (27 to 53%) and significant reductions in leaf weight (40 to 67%) and shoot weight (33 to 60%) after being stem punctured into Gossypium hirsutum 'Rowden'. They caused little or no significant reductions in shoot weight and height or increases in foliar symptoms and vascular browning in a soil-infestation assay. Soil infestation with Meloidogyne incognita race 3 (root-knot nematode) alone also failed to cause significant disease. When coinoculated with M. incognita race 3, all VCG caused moderate to severe wilt. Therefore, the VCG identified in this study belong to the vascular-competent pathotype, and should pose similar threats to cotton cultivars in the presence of the root-knot nematode. Use of nematode-resistant cultivars, therefore, is probably the best approach to control the disease in Georgia.

Fine mapping and identification of candidate genes for a QTL affecting Meloidogyne incognita reproduction in Upland cotton.

BACKGROUND: The southern root-knot nematode (Meloidogyne incognita; RKN) is one of the most important economic pests of Upland cotton (Gossypium hirsutum L.). Host plant resistance, the ability of a plant to suppress nematode reproduction, is the most economical, practical, and environmentally sound method to provide protection against this subterranean pest. The resistant line Auburn 623RNR and a number of elite breeding lines derived from it remain the most important source of root-knot nematode (RKN) resistance. Prior genetic analysis has identified two epistatically interacting RKN resistance QTLs, qMi-C11 and qMi-C14, affecting gall formation and RKN reproduction, respectively. RESULTS: We developed a genetic population segregating only for the qMi-C14 locus and evaluated the genetic effects of this QTL on RKN resistance in the absence of the qMi-C11 locus. The qMi-C14 locus had a LOD score of 12 and accounted for 24.5 % of total phenotypic variation for egg production. In addition to not being significantly associated with gall formation, this locus had a lower main effect on RKN reproduction than found in our previous study, which lends further support to evidence of epistasis with qMi-C11 in imparting RKN resistance in the Auburn 623RNR source. The locus qMi-C14 was fine-mapped with the addition of 16 newly developed markers. By using the reference genome sequence of G. raimondii, we identified 20 candidate genes encoding disease resistance protein homologs in the newly defined 2.3 Mb region flanked by two SSR markers. Resequencing of an RKN resistant and susceptible G. hirsutum germplasm revealed non-synonymous mutations in only four of the coding regions of candidate genes, and these four genes are consequently of high interest. CONCLUSIONS: Our mapping results validated the effects of the qMi-C14 resistance locus, delimiting the QTL to a smaller region, and identified tightly linked SSR markers to improve the efficiency of marker-assisted selection. The candidate genes identified warrant functional studies that will help in identifying and characterizing the actual qMi-C14 defense gene(s) against root-knot nematodes.