Distribution of Three Verticillium dahliae Races in Coastal California and Evaluation of Resistance in Lettuce.

Verticillium wilt, caused by Verticillium dahliae, is one of the most devastating soilborne diseases of lettuce (Lactuca sativa L.). There are three races of V. dahliae, and each race has been characterized by markers representing race-specific effectors. Race 1 is differentiated by the presence of the functional secretory Ave1 effector. Similarly, races 2 and 3 are differentiated by effectors VdR2e and VdR3e, respectively. Although the presence of race 1 in coastal California was well established, the presence of effector-based races 2 and 3 was uncertain. This study therefore focused on characterizing 727 isolates collected from 142 ranches of symptomatic lettuce and other crops from coastal California. Based on this evaluation, 523 isolates were designated as race 1, 20 isolates as race 2, 23 isolates as race 3, and 17 as race undefined. Isolates representing other Verticillium species totaled 110, and 34 were non-Verticillium fungal species. Because the use of resistant cultivars is a key strategy to manage this disease, we evaluated 48 lettuce germplasm lines and 1 endive (Cichorium endivia L.) line, comprising commercial cultivars and breeding lines, including the race 1-resistant heirloom cultivar La Brillante and the susceptible cultivar Salinas as controls. Resistance against races 1, 2, and 3 along with VdLs17, a virulent isolate of V. dahliae from lettuce that is currently not assigned to a race, was evaluated in replicated greenhouse experiments. Two crisphead lettuce lines, HL28 and HL29, exhibited resistance against race 1 and a partial resistance against race 2, whereas all other lines were highly susceptible to races 1 and 2 and VdLs17. The majority of lines exhibited higher resistance to race 3 relative to the other two races. This study documents the current distribution of the different races in coastal California. In addition, the sources of resistance currently being developed should be effective or partially effective against these races for targeted deployment as soon as they are available.

Examining preharvest genetic and morphological factors contributing to lettuce (Lactuca sativa L.) shelf-life.

Lettuce is a highly perishable horticultural crop with a relatively short shelf-life that limits its commercial value and contributes to food waste. Postharvest senescence varies with influences of both environmental and genetic factors. From a larger pool of romaine lettuce genotypes, we identified three genotypes with variable shelf lives and evaluated their leaf morphology characteristics and transcriptomic profiles at preharvest to predict postharvest quality. Breeding line 60184 had the shortest shelf-life (SSL), cultivar 'Manatee' had an intermediate shelf-life (ISL), and 'Okeechobee' had the longest shelf-life (LSL). We observed significantly larger leaf lamina thickness and higher stomatal index in the SSL genotypes relative to the LSL cultivar. To identify molecular indicators of shelf-life, we used a transcriptional approach between two of the contrasting genotypes, breeding line 60184 and cultivar 'Okeechobee' at preharvest. We identified 552 upregulated and 315 downregulated differentially expressed genes between the genotypes, from which 27% of them had an Arabidopsis thaliana ortholog previously characterized as senescence associated genes (SAGs). Notably, we identified several SAGs including several related to jasmonate ZIM-domain jasmonic acid signaling, chlorophyll a-b binding, and cell wall modification including pectate lyases and expansins. This study presented an innovative approach for identifying preharvest molecular factors linked to postharvest traits for prolonged shelf.

Identification of Quantitative Trait Loci Associated with Bacterial Leaf Spot Resistance in Baby Leaf Lettuce.

Spring mix is a popular packaged salad that contains lettuce (Lactuca sativa L.) as one of its main ingredients. Plants for baby leaf lettuce (BLL) production are grown at very high densities, which enhances the occurrence of bacterial leaf spot (BLS) caused by Xanthomonas hortorum pv. vitians (Xhv), a disease that can make the crop unmarketable. The market demands disease-free, high-quality BLL all year round. Growing highly BLS-resistant cultivars will reduce loss of yield and quality, thus minimizing economic detriment to lettuce and spring mix growers. The research objectives were to identify lettuce accessions resistant to BLS and associated quantitative trait loci (QTL). A total of 495 lettuce accessions were screened with six isolates (BS0347, BS2861, BS3127, L7, L44, and Sc8B) of Xhv. Accessions showing overall high-level resistance to all tested Xhv isolates were 'Bunte Forellen', PI 226514, 'La Brillante', ARM09-161-10-1-4, 'Grenadier', 'Bella', PI 491210, 'Delight', and 'Romana Verde del Mercado'. Genome-wide association studies of BLS resistance by mixed linear model analyses identified significant QTLs on four lettuce chromosomes (2, 4, 6, and 8). The most significant QTL was on Chromosome 8 (P = 1.42 × 10-7), which explained 6.7% of total phenotypic variation for the disease severity. Accessions with a high level of resistance detected in this study are valuable resources for lettuce germplasm improvement. Molecular markers closely linked to QTLs can be considered for marker-assisted selection to develop new BLL lettuce cultivars with resistance to multiple races of Xhv.

Identification and mapping of quantitative trait loci for resistance to Liriomyza trifolii in romaine lettuce cultivar 'Valmaine'.

Liriomyza trifolii (Diptera: Agromyzidae) is a leafminer that causes ruinous damage to many leafy vegetables including lettuce (Lactuca sativa L.) by stippling and tunneling the leaves. In this study, a population of 125 F3 families was developed from the intraspecific cross of 'Valmaine' (resistant) and 'Okeechobee' (susceptible) romaine cultivars for inheritance analysis and molecular mapping of the resistance loci controlling stippling damage. The experiments were conducted in an insectarium (controlled environment). Stippling damage proved to be heritable because the broad-sense heritability (H2) was 0.58. A segregation analysis suggested that a single dominant allele, Sd1 locus, controls resistance against L. trifolii. Furthermore, a quantitative trait loci (QTL) analysis identified one novel QTL, named Stippling on LG5 (qSTP5), flanked by two SNPs that were mapped to a 5.2 cM (8.5 Mb region) interval, explaining over 13% of the total phenotypic variance. Desirable allele for resistance to L. trifolii was derived from resistant cultivar Valmaine. Identification of SNPs closely linked to the QTL responsible for L. trifolii resistance should facilitate plant breeders to develop resistant romaine lettuce cultivars.

Genetics of Partial Resistance Against Verticillium dahliae Race 2 in Wild and Cultivated Lettuce.

Lettuce (Lactuca sativa) is one of the most economically important vegetables in the United States, with approximately 50% of the domestic production concentrated in the Salinas Valley of California. Verticillium wilt, caused by races 1 and 2 of the fungal pathogen Verticillium dahliae, poses a major threat to lettuce production in this area. Although resistance governed by a single dominant gene against race 1 has previously been identified and is currently being incorporated into commercial cultivars, identification of resistance against race 2 has been challenging and no lines with complete resistance have been identified. In this study, we screened germplasm for resistance and investigated the genetics of partial resistance against race 2 using three mapping populations derived from crosses involving L. sativa × L. sativa and L. serriola × L. sativa. The inheritance of resistance in Lactuca species against race 2 is complex but a common quantitative trait locus (QTL) on linkage group 6, designated qVERT6.1 (quantitative Verticillium dahliae resistance on LG 6, first QTL), was detected in multiple populations. Additional race 2 resistance QTLs located in several linkage groups were detected in individual populations and environments. Because resistance in lettuce against race 2 is polygenic with a large genotype by environment interaction, breeding programs to incorporate these resistance genes should be aware of this complexity as they implement strategies to control race 2.

Short-Term Host Selection Pressure Has Little Effect on the Evolution of a Monoclonal Population of Verticillium dahliae Race 1.

Understanding pathogen evolution over time is vital for plant breeding and deployment of host resistance. In the context of a soilborne pathogen, the potential of host-directed evolution of a Verticillium dahliae race 1 isolate and genotypic variation of V. dahliae associated with two major hosts (lettuce and tomato) were determined. In total, 427 isolates were recovered over 6 years from a resistance screening nursery infested with a single V. dahliae race 1 isolate. In a separate study, an additional 206 isolates representing 163 and 43 isolates from commercial lettuce and tomato fields, respectively, were collected. Analyses of isolates recovered from the screening nursery over 6 years revealed no changes in the race and mating type composition but did uncover seven simple sequence repeat (SSR) variant genotypes. No significant genotypic variation in V. dahliae was observed between or within fields of either lettuce or tomato but pathogen populations were significantly differentiated between these two hosts. Replicated virulence assays of variant SSR genotypes on lettuce differential cultivars suggested no significant difference in virulence from the wild-type race 1 isolate introduced into the field. This suggests that deployed race 1 host resistance will be robust against the widespread race 1 populations in lettuce-growing regions at least for 6 years unless novel pathogen genotypes or races are introduced into the system.

Defensive changes in maize leaves induced by feeding of Mediterranean corn borer larvae.

BACKGROUND: Plants can respond to insect attack via defense mechanisms that reduce insect performance. In this study, we examined the effects of several treatments applied to two maize genotypes (one resistant, one susceptible) on the subsequent growth and survival of Sesamia nonagrioides Lef. (Mediterranean corn borer, MCB) larvae. The treatments were infestation with MCB larvae, application of MCB regurgitant upon wounding, wounding alone, or exposure to methyl jasmonate, and they were applied at the V6-V8 stage of maize development. We also monitored changes in the concentrations of compounds known to be involved in constitutive resistance, such as cell wall-bound hydroxycinnamates and benzoxazinoids. RESULTS: In both maize genotypes, the leaves of plants pre-infested with MCB larvae were less suitable for larval development than those from untreated plants. Application of MCB regurgitant upon wounding, and wounding itself, resulted in leaf tissues becoming less suitable for larval growth than those of pre-infested plants, suggesting that there could be herbivore-associated effector molecules that suppress some wounding responses. A single application of MCB regurgitant did not seem to mimic feeding by MCB larvae, although the results suggested that regurgitant deposited during feeding may have enhanced ferulates and diferulates synthesis in infested vs. control plants. Jasmonic acid may play a role in mediating the maize response to MCB attack, but it did not trigger hydroxycinnamate accumulation in the leaves to a level comparable to that induced by larval leaf feeding. The EP39 maize genotype showed an increase in leaf cell wall strength by increasing hemicellulose cross-linking in response to MCB attack, while induced defenses in the EP42 plants appeared to reflect a broader array of resistance mechanisms. CONCLUSIONS: The results indicated that leaf feeding by MCB larvae can increase leaf antibiosis against MCB in two maize genotypes with contrasting levels of resistance against this borer. Also, the larval regurgitant played a positive role in eliciting a defense response. We determined the effects of the plant response on larval growth, and detected defense compounds related to borer resistance.

Dynamics of verticillium species microsclerotia in field soils in response to fumigation, cropping patterns, and flooding.

Verticillium dahliae is a soilborne, economically significant fungal plant pathogen that persists in the soil for up to 14 years as melanized microsclerotia (ms). Similarly, V. longisporum is a very significant production constraint on members of the family Brassicaceae. Management of Verticillium wilt has relied on methods that reduce ms below crop-specific thresholds at which little or no disease develops. Methyl bromide, a broad-spectrum biocide, has been used as a preplant soil fumigant for over 50 years to reduce V. dahliae ms. However, reductions in the number of ms in the vertical and horizontal soil profiles and the rate at which soil recolonization occurs has not been studied. The dynamics of ms in soil before and after methyl bromide+chloropicrin fumigation were followed over 3 years in six 8-by-8-m sites in two fields. In separate fields, the dynamics of ms in the 60-cm-deep vertical soil profile pre- and postfumigation with methyl bromide+chloropicrin followed by various cropping patterns were studied over 4 years. Finally, ms densities were assessed in six 8-by-8-m sites in a separate field prior to and following a natural 6-week flood. Methyl bromide+chloripicrin significantly reduced but did not eliminate V. dahliae ms in either the vertical or horizontal soil profiles. In field studies, increases in ms were highly dependent upon the crop rotation pattern followed postfumigation. In the vertical soil profile, densities of ms were highest in the top 5 to 20 cm of soil but were consistently detected at 60-cm depths. Six weeks of natural flooding significantly reduced (on average, approximately 65% in the total viable counts of ms) but did not eliminate viable ms of V. longisporum.

The Three Lineages of the Diploid Hybrid Verticillium longisporum Differ in Virulence and Pathogenicity.

Verticillium longisporum is an economically important vascular pathogen of Brassicaceae crops in different parts of the world. V. longisporum is a diploid hybrid that consists of three different lineages, each of which originated from a separate hybridization event between two different sets of parental species. We used 20 isolates representing the three V. longisporum lineages and the relative V. dahliae, and performed pathogenicity tests on 11 different hosts, including artichoke, cabbage, cauliflower, cotton, eggplant, horseradish, lettuce, linseed, oilseed rape (canola), tomato, and watermelon. V. longisporum was overall more virulent on the Brassicaceae crops than V. dahliae, which was more virulent than V. longisporum across the non-Brassicaceae crops. There were differences in virulence between the three V. longisporum lineages. V. longisporum lineage A1/D1 was the most virulent lineage on oilseed rape, and V. longisporum lineage A1/D2 was the most virulent lineage on cabbage and horseradish. We also found that on the non-Brassicaceae hosts eggplant, tomato, lettuce, and watermelon, V. longisporum was more or equally virulent than V. dahliae. This suggests that V. longisporum may have a wider potential host range than currently appreciated.

Host Range of Verticillium isaacii and Verticillium klebahnii from Artichoke, Spinach, and Lettuce.

Verticillium is a genus that includes major vascular wilt pathogens. Recently, multilocus phylogenetic analyses of the genus identified five new species, including Verticillium isaacii and V. klebahnii, both of which occur in agricultural soils in coastal California and have been isolated from asymptomatic and diseased spinach and lettuce plants. Little data are available regarding their pathogenicity and virulence on a broader range of crops important to the region. Four isolates each of V. isaacii and V. klebahnii along with two reference isolates of V. dahliae races 1 and 2 were inoculated on eight crops (artichoke, cauliflower, eggplant, lettuce, pepper, tomato, spinach, and strawberry) in a greenhouse experiment. After 8 weeks, plants were assessed for disease severity to determine the relative host ranges of Verticillium isolates. Additionally, 13 lettuce lines resistant to race 1 and partially resistant to race 2 of V. dahliae were screened against V. isaacii and V. klebahnii to evaluate their responses. Three of four V. isaacii and four of four V. klebahnii isolates tested were nonpathogenic on all crops tested except those indicated below. One V. isaacii isolate caused wilt on artichoke and 'Salinas' lettuce and most isolates of both species caused varying degrees of Verticillium wilt on strawberry. Lettuce lines resistant to V. dahliae race 1 and partially resistant to V. dahliae race 2 also exhibited resistance to all of the isolates of V. isaacii and V. klebahnii. Thus, at least some isolates in the populations of V. isaacii and V. klebahnii have the potential to become significant pathogens of coastal California crops. However, resistance developed against V. dahliae also offers resistance to the pathogenic isolates of both species, at least in lettuce.

Screening of Wild and Cultivated Capsicum Germplasm Reveals New Sources of Verticillium Wilt Resistance.

Verticillium wilt caused by Verticillium dahliae is an important soilborne disease of pepper (Capsicum species) worldwide. Most commercial pepper cultivars lack resistance to this pathogen. Our objective was to identify resistance to two V. dahliae isolates in wild and cultivated Capsicum accessions from the core collection of the National Plant Germplasm System of the USDA. Screening of 397 Capsicum accessions against two V. dahliae isolates (Vdca59 and VdCf45) was performed in a greenhouse. Seventy-eight accessions selected from this screen were further evaluated in a follow-up experiment. In total, 21 (26.9%) and 13 (16.6%) Capsicum accessions tested were resistant to Verticillium wilt when inoculated with V. dahliae isolates VdCa59 and VdCf45, respectively. Eight accessions (Grif 9073, PI 281396, PI 281397, PI 438666, PI 439292, PI 439297, PI 555616, and PI 594125) were resistant to Verticillium wilt against both V. dahliae isolates. On the basis of Germplasm Resources Information Network data, two of the Capsicum annuum accessions (Grif 9073 and PI 439297) were also resistant to Phytophthora root rot disease. These sources of multiple disease resistance will be useful to pepper breeding programs.

Nondefoliating and Defoliating Strains from Cotton Correlate with Races 1 and 2 of Verticillium dahliae.

Verticillium wilt, caused by Verticillium dahliae, is an important disease of cotton worldwide. Isolates of V. dahliae can be characterized as race 1 or race 2 based on the responses of differential cultivars of tomato and lettuce, or as defoliating or nondefoliating based on symptom expression in cotton. To investigate the frequency and distribution of races and defoliation phenotypes of cotton-associated V. dahliae, 317 isolates from China, Israel, Turkey, and the United States were tested by polymerase chain reaction (PCR) using defoliating, nondefoliating, and race 1- and race 2-specific primers DF/DR, NDF/NDR, VdAve1F/VdAve1R, and VdR2F/VdR2R, respectively. Of the total, 97.2% of isolates genotyped as defoliating were also characterized as race 2, while 90.8% of isolates genotyped as nondefoliating were also genotyped as race 1. To verify these results, three cotton cultivars-'FM 2484B2F' (highly resistant), '98M-2983' (highly susceptible), and 'CA4002' (partially resistant)-used as differentials were each inoculated with 10 isolates characterized by PCR: six defoliating/race 2 strains (GH1005, GH1021, HN, XJ2008, XJ592, and reference strain Ls17) and four nondefoliating/race 1 strains (GH1015, GH1016, GH1020, and reference strain Ls16). All defoliating/race 2 isolates except for Ls17 caused defoliation on 98M-2983 and CA4002. Isolate Ls17 caused defoliation on 98M-2983 only. The nondefoliating/race 1 isolates caused Verticillium wilt symptoms devoid of defoliation on 98M-2983. The greenhouse assays confirmed the molecular identification of race and defoliation phenotype. Although the existence of races has not been previously established among V. dahliae isolates from cotton, the long-established nondefoliating and defoliating population structure corresponded with V. dahliae races 1 and 2, respectively.

Maize toxin degrades peritrophic matrix proteins and stimulates compensatory transcriptome responses in fall armyworm midgut.

Understanding the molecular mechanisms underlying insect compensatory responses to plant defenses could lead to improved plant resistance to herbivores. The Mp708 inbred line of maize produces the maize insect resistant 1-cysteine protease (Mir1-CP) toxin. Reduced feeding and growth of fall armyworm larvae fed on Mp708 was previously linked to impairment of nutrient utilization and degradation of the midgut (MG) peritrophic matrix (PM) by Mir1-CP. Here we examine the biochemical and transcriptional responses of fall armyworm larvae to Mir1-CP. Insect Intestinal Mucin (IIM) was severely depleted from pure PMs treated in vitro with recombinant Mir1-CP. Larvae fed on Mp708 midwhorls excrete frass largely depleted of IIM. Cracks, fissures and increased porosity previously observed in the PM of larvae fed on Mp708 midwhorls could ensue when Mir1-CP degrades the IIM that cross-links chitin fibrils in the PM. Both targeted and global transcriptome analyses were performed to determine how complete dissolution of the structure and function of the PM is prevented, enabling larvae to continue growing in the presence of Mir1-CP. The MGs from fall armyworm fed on Mp708 upregulate expression of genes encoding proteins involved in PM production as an apparent compensation to replace the disrupted PM structure and restore appropriate counter-current MG gradients. Also, several families of digestive enzymes (endopeptidases, aminopeptidases, lipases, amylase) were more highly expressed in MGs from larvae fed on Mp708 than MGs from larvae fed on diets lacking Mir1-CP (artificial diet, midwhorls from Tx601 or B73 maize). Impaired growth of larvae fed on Mp708 probably results from metabolic costs associated with higher production of PM constituents and digestive enzymes in a compensatory attempt to maintain MG function.

Belowground resistance to western corn rootworm in lepidopteran-resistant maize genotypes.

Several maize, Zea mays L., inbred lines developed from an Antiguan maize population have been shown to exhibit resistance to numerous aboveground lepidopteran pests. This study shows that these genotypes are able to significantly reduce the survival of two root feeding pests, western corn rootworm, Diabrotica virgifera virgifera LeConte, and southern corn rootworm, Diabrotica undecimpunctata howardi Barber. The results also demonstrated that feeding by the aboveground herbivore fall armyworm, Spodoptera frugiperda (J. E. Smith), before infestation by western corn rootworm reduced survivorship of western corn rootworm in the root tissues of some, but not all, genotypes. Likewise, the presence of western corn rootworm in the soil seemed to increase resistance to fall armyworm in the whorl in several genotypes. However, genotypes derived from the Antiguan germplasm with genetic resistance to lepidopterans were still more resistant to the fall armyworm and both rootworm species than the susceptible genotypes even after defense induction. These results suggest that there may be intraplant communication that alters plant responses to aboveground and belowground herbivores.

Mapping of QTL for resistance to the Mediterranean corn borer attack using the intermated B73 x Mo17 (IBM) population of maize.

The Mediterranean corn borer or pink stem borer (MCB, Sesamia nonagrioides Lefebvre) causes important yield losses as a consequence of stalk tunneling and direct kernel damage. B73 and Mo17 are the source of the most commercial valuable maize inbred lines in temperate zones, while the intermated B73 x Mo17 (IBM) population is an invaluable source for QTL identification. However, no or few experiments have been carried out to detect QTL for corn borer resistance in the B73 x Mo17 population. The objective of this work was to locate QTL for resistance to stem tunneling and kernel damage by MCB in the IBM population. We detected a QTL for kernel damage at bin 8.05, although the effect was small and two QTL for stalk tunneling at bins 1.06 and 9.04 in which the additive effects were 4 cm, approximately. The two QTL detected for MCB resistance were close to other QTL consistently found for European corn borer (ECB, Ostrinia nubilalis Hübner) resistance, indicating mechanisms of resistance common to both pests or gene clusters controlling resistance to different plagues. The precise mapping achieved with the IBM population will facilitate the QTL pyramiding and the positional cloning of the detected QTL.

Changes in phenolic concentrations during recurrent selection for resistance to the Mediterranean corn borer (Sesamia nonagrioides Lef.).

Recurrent selection has been reported as successful for improving maize resistance against corn borers. This study was conducted to determine if phenolics concentration in maize changes during recurrent selection to improve stalk resistance to the Mediterranean corn borer. Three cycles of selection [EPS12(S)C0, ESP12(S)C2, and EPS12(S)C3] from the maize synthetic population EPS12 and test crosses to inbred lines A639, B93, and EP42 were field grown and artificially infested with Mediterranean corn borer larvae, and the pith tissues were sampled for biochemical analyses. Two major simple phenolic acids [p-coumaric (p-CA) and trans-ferulic (E-FA) acids] were identified in free and cell-wall fractions, whereas four isomers of diferulic acid (DFA) (8-5'l, 5-5', 8-o-4', and 8-5' benzofuran form) were present in the cell-wall bound fraction. The selection cycles EPS12(S)C0 and EPS12(S)C3 showed less damage and higher cell wall phenolics concentrations than the cycle EPS12(S)C2. In addition, higher concentrations of total DFAs were associated with shorter tunnel length and lower numbers of larvae per stem. The current study shows new and concrete evidence that the cell-wall bound phenolics could have a determinative role in the resistance to the Mediterranean corn borer, although future development of recurrent and divergent selection cycles will clarify this point.

Diferulate content of maize sheaths is associated with resistance to the Mediterranean corn borer Sesamia nonagrioides (Lepidoptera: Noctuidae).

The leaf sheaths of selected inbred lines of maize (Zea mays L.) with variable levels of stem resistance to the Mediterranean corn borer Sesamia nonagrioides (Lefèvbre) were evaluated for antibiotic effect on insect development. Phytochemical analyses of leaf sheaths were conducted for cell wall phenylpropanoid content to gain a better understanding of maize-resistance mechanisms. Laboratory bioassays established that sheath tissues from different genotypes significantly affected the growth of neonate larvae. Three hydroxycinnamates, p-coumaric, trans-ferulic, and cis-ferulic acids, and three isomers of diferulic acid, 8-5', 8-O-4', and 8-5' b (benzofuran form), were identified. Significant negative correlations were found between larvae weight and diferulic acid content for six genotypes. These results are in agreement with previous studies concerning the role of cell wall structural components in stem borer resistance.