Comparison of assembly platforms for the assembly of the nuclear genome of Trichoderma harzianum strain PAR3.

BACKGROUND: Trichoderma is a diverse genus of fungi that includes several species that possess biotechnological and agricultural applications, including the biocontrol of pathogenic fungi and nematodes. The mitochondrial genome of a putative strain of Trichoderma harzianum called PAR3 was analyzed after isolation from the roots of Scarlet Royal grapevine scion grafted to Freedom rootstock, located in a grapevine vineyard in Parlier, CA, USA. Here, we report the sequencing, comparative assembly, and annotation of the nuclear genome of PAR3 and confirm its identification as a strain of T. harzianum. We subsequently compared the genes found in T. harzianum PAR3 to other known T. harzianum strains. Assembly of Illumina and/or Oxford Nanopore reads by the popular long-read assemblers, Flye and Canu, and the hybrid assemblers, SPAdes and MaSuRCA, was performed and the quality of the resulting assemblies were compared to ascertain which assembler generated the highest quality draft genome assembly. RESULTS: MaSuRCA produced the most complete and high-fidelity assembly yielding a nuclear genome of 40.7 Mb comprised of 112 scaffolds. Subsequent annotation of this assembly produced 12,074 gene models and 210 tRNAs. This included 221 genes that did not have equivalent genes in other T. harzainum strains. Phylogenetic analysis of ITS, rpb2, and tef1a sequences from PAR3 and established Trichoderma spp. showed that all three sequences from PAR3 possessed more than 99% identity to those of Trichoderma harzianum, confirming that PAR3 is an isolate of Trichoderma harzianum. We also found that comparison of gene models between T. harzianum PAR3 and other T. harzianum strains resulted in the identification of significant differences in gene type and number, with 221 unique genes identified in the PAR3 strain. CONCLUSIONS: This study gives insight into the efficacy of several popular assembly platforms for assembly of fungal nuclear genomes, and found that the hybrid assembler, MaSuRCA, was the most effective program for genome assembly. The annotated draft nuclear genome and the identification of genes not found in other T. harzainum strains could be used to investigate the potential applications of T. harzianum PAR3 for biocontrol of grapevine fungal canker pathogens and as source of anti-microbial compounds.

Overexpression of maize ZmLOX6 in Arabidopsis thaliana enhances damage-induced pentyl leaf volatile emissions that affect plant growth and interaction with aphids.

Pentyl leafy volatiles (PLV) are C5 volatiles produced from polyunsaturated fatty acids by plant 13-lipoxygenases (13-LOX) in concert with other lipid metabolizing enzymes. Unlike related C6 volatiles (GLV, green leafy volatiles), little is known about the biosynthesis and physiological function of PLV in plants. Zea mays LOX6 (ZmLOX6) is an unusual plant LOX that lacks lipid oxygenation activity but acts as a hydroperoxide lyase hypothesized to be specifically involved in PLV synthesis. We overexpressed ZmLOX6 in Arabidopsis thaliana and established that it indeed produces PLVs. Overexpression of ZmLOX6 caused a mild chlorotic phenotype, and induced a similar phenotype in untransformed Col-0 plants grown in close proximity, suggesting that airborne signals, such as PLVs, are responsible for the phenotype. PLV production, dependency on the substrate from endogenous 13-LOX(s), and likely competition with endogenous 13-oxylipin pathway were consistent with the model that ZmLOX6 functions as a hydroperoxide lyase. The abundance of individual PLVs was differentially affected by ZmLOX6 overexpression, and the new profile indicated that ZmLOX6 had reaction products distinct from endogenous PLV-producing activities in the Arabidopsis host plants. ZmLOX6 overexpression also induced a new hormonal status, which is likely responsible for increased attraction and propagation of aphids, nonetheless improving host plant tolerance to aphid infestation.

Pre-inoculation water deficit effects on grapevine physiology, Xylella fastidiosa titers, and Pierce's disease progression.

Drought and Pierce's disease are common throughout many grapevine-growing regions such as Mexico and the United States. Yet, how ongoing water deficits affect infections of Xylella fastidiosa, the causal agent of Pierce's disease, is poorly understood. Symptoms were observed to be significantly more severe in water-stressed plants one month after X. fastidiosa inoculation, and, in one experiment, titers were significantly lower in water-stressed than well-watered grapevines. Host chemistry examinations revealed overall amino acid and phenolic levels did not statistically differ due to water deficits, but sugar levels were significantly greater in water stressed than well-watered plants. Results highlight the need to especially manage Pierce's disease spread in grapevines experiencing drought.

Cytokinin Promotes Jasmonic Acid Accumulation in the Control of Maize Leaf Growth.

Plant organ growth results from the combined activity of cell division and cell expansion. The co-ordination of these two processes depends on the interplay between multiple hormones that determine the final organ size. Using the semidominant Hairy Sheath Frayed1 (Hsf1) maize mutant that hypersignals the perception of cytokinin (CK), we show that CK can reduce leaf size and growth rate by decreasing cell division. Linked to CK hypersignaling, the Hsf1 mutant has an increased jasmonic acid (JA) content, a hormone that can inhibit cell division. The treatment of wild-type seedlings with exogenous JA reduces maize leaf size and growth rate, while JA-deficient maize mutants have increased leaf size and growth rate. Expression analysis revealed the increased transcript accumulation of several JA pathway genes in the Hsf1 leaf growth zone. A transient treatment of growing wild-type maize shoots with exogenous CK also induced the expression of JA biosynthetic genes, although this effect was blocked by the co-treatment with cycloheximide. Together, our results suggest that CK can promote JA accumulation, possibly through the increased expression of specific JA pathway genes.

9,10-KODA, an α-ketol produced by the tonoplast-localized 9-lipoxygenase ZmLOX5, plays a signaling role in maize defense against insect herbivory.

13-Lipoxygenases (LOXs) initiate the synthesis of jasmonic acid (JA), the best-understood oxylipin hormone in herbivory defense. However, the roles of 9-LOX-derived oxylipins in insect resistance remain unclear. Here, we report a novel anti-herbivory mechanism mediated by a tonoplast-localized 9-LOX, ZmLOX5, and its linolenic acid-derived product, 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid (9,10-KODA). Transposon-insertional disruption of ZmLOX5 resulted in the loss of resistance to insect herbivory. lox5 knockout mutants displayed greatly reduced wound-induced accumulation of multiple oxylipins and defense metabolites, including benzoxazinoids, abscisic acid (ABA), and JA-isoleucine (JA-Ile). However, exogenous JA-Ile failed to rescue insect defense in lox5 mutants, while applications of 1 µM 9,10-KODA or the JA precursor, 12-oxo-phytodienoic acid (12-OPDA), restored wild-type resistance levels. Metabolite profiling revealed that exogenous 9,10-KODA primed the plants for increased production of ABA and 12-OPDA, but not JA-Ile. While none of the 9-oxylipins were able to rescue JA-Ile induction, the lox5 mutant accumulated lower wound-induced levels of Ca2+, suggesting this as a potential explanation for lower wound-induced JA. Seedlings pretreated with 9,10-KODA exhibited rapid or more robust wound-induced defense gene expression. In addition, an artificial diet supplemented with 9,10-KODA arrested fall armyworm larvae growth. Finally, analysis of single and double lox5 and lox10 mutants showed that ZmLOX5 also contributed to insect defense by modulating ZmLOX10-mediated green leaf volatile signaling. Collectively, our study uncovered a previously unknown anti-herbivore defense and hormone-like signaling activity for a major 9-oxylipin α-ketol.

Amino acid, sugar, phenolic, and terpenoid profiles are capable of distinguishing Citrus tristeza virus infection status in citrus cultivars: Grapefruit, lemon, mandarin, and sweet orange.

Citrus tristeza virus (CTV) is the most severe viral disease for citrus production. Many strains of CTV have been characterized and their symptomology widely varies, ranging from asymptomatic or mild infections to severe symptomology that results in substantial yield loss or host death. The capacity of the different CTV strains to affect the biochemistry of different citrus species has remained largely unstudied, despite that associated metabolomic shifts would be relevant toward symptom development. Thus, amino acid, sugar, phenolic, and terpenoid levels were assessed in leaves of healthy and CTV-infected grapefruit, lemon, mandarin, and two different sweet orange cultivars. Both mild [VT-negative (VT-)] and severe [VT-positive (VT+)] CTV genotype strains were utilized. When looking at overall totals of these metabolite classes, only amino acid levels were significantly increased by infection of citrus with severe CTV strains, relative to mild CTV strains or healthy plants. No significant trends of CTV infection on summed amounts of all sugar, phenolic, or terpenoid compounds were observed. However, individual compound levels were affected by CTV infections. Subsequent canonical discriminant analysis (CDA) that utilized profiles of individual amino acids, terpenoids, or phenolics successfully distinguished leaf samples to specific citrus varieties and identified infection status with good accuracy. Collectively, this study reveals biochemical patterns associated with severity of CTV infections that can potentially be utilized to help identify in-field CTV infections of economic relevance.

Mixed infections of fungal trunk pathogens and induced systemic phenolic compound production in grapevines.

As grapevines mature in California vineyards they accumulate chronic wood infections by the Ascomycete fungi that cause trunk diseases, including Botryosphaeria dieback (caused by Diplodia seriata and Neofusicoccum parvum) and Esca (caused by Phaeomoniella chlamydospora). It is thought that such mixed infections become localized to separate internal lesions/cankers of the permanent, woody structure of an individual vine, but nonetheless the fungi all colonize the same vascular system. In response to infection by one pathogen, the host may initiate systemic biochemical changes, which in turn may affect the extent of subsequent infections by other pathogens. To test this hypothesis, we measured changes in phenolic compounds in the wood and lesion lengths of the pathogens, during sequential co-inoculations with different or identical pair-wise sequences of infection by D. seriata, N. parvum, or P. chlamydospora. Prior fungal infections only affected the development of subsequent D. seriata infections. Effects of fungal infections on phenolic compounds were variable, yet initial infection by D. seriata was associated with significantly higher concentrations of most phenolic compounds distally, compared to all other initial inoculation treatments. It was hypothesized that pre-existing phenolic levels can slow initial lesion development of fungal trunk pathogens, especially for D. seriata, but over time the pathogens appeared to overcome or neutralize phenolic compounds and grow unimpeded. These results demonstrate that effects of one fungal trunk pathogen infection is generally unable to distally affect another long-term, albeit shifts in host phenolics and other plant defenses do occur.

Trehalose increases tomato drought tolerance, induces defenses, and increases resistance to bacterial wilt disease.

Ralstonia solanacearum causes bacterial wilt disease, leading to severe crop losses. Xylem sap from R. solanacearum-infected tomato is enriched in the disaccharide trehalose. Water-stressed plants also accumulate trehalose, which increases drought tolerance via abscisic acid (ABA) signaling. Because R. solanacearum-infected plants suffer reduced water flow, we hypothesized that bacterial wilt physiologically mimics drought stress, which trehalose could mitigate. We found that R. solanacearum-infected plants differentially expressed drought-associated genes, including those involved in ABA and trehalose metabolism, and had more ABA in xylem sap. Consistent with this, treating tomato roots with ABA reduced both stomatal conductance and stem colonization by R. solanacearum. Treating roots with trehalose increased xylem sap ABA and reduced plant water use by lowering stomatal conductance and temporarily improving water use efficiency. Trehalose treatment also upregulated expression of salicylic acid (SA)-dependent tomato defense genes; increased xylem sap levels of SA and other antimicrobial compounds; and increased bacterial wilt resistance of SA-insensitive NahG tomato plants. Additionally, trehalose treatment increased xylem concentrations of jasmonic acid and related oxylipins. Finally, trehalose-treated plants were substantially more resistant to bacterial wilt disease. Together, these data show that exogenous trehalose reduced both water stress and bacterial wilt disease and triggered systemic disease resistance, possibly through a Damage Associated Molecular Pattern (DAMP) response pathway. This suite of responses revealed unexpected linkages between plant responses to biotic and abiotic stress and suggested that R. solanacearum-infected plants increase trehalose to improve water use efficiency and increase wilt disease resistance. The pathogen may degrade trehalose to counter these efforts. Together, these results suggest that treating tomatoes with exogenous trehalose could be a practical strategy for bacterial wilt management.

The Synthesis of Pentyl Leaf Volatiles and Their Role in Resistance to Anthracnose Leaf Blight.

Volatiles are important airborne chemical messengers that facilitate plant adaptation to a variety of environmental challenges. Lipoxygenases (LOXs) produce a bouquet of non-volatile and volatile oxylipins, including C6 green leaf volatiles (GLVs), which are involved in a litany of plant physiological processes. GLVs are emitted by a diverse array of plant species, and are the best-known group of LOX-derived volatiles. Five-carbon pentyl leaf volatiles (PLVs) represent another widely emitted group of LOX-derived volatiles that share structural similarity to GLVs, however, relatively little is known about their biosynthesis or biological activity. In this study, we utilized PLV-deficient mutants of maize and Arabidopsis and exogenous PLV applications to elucidate the biosynthetic order of individual PLVs. We further measured PLVs and GLVs after tissue disruption of leaves by two popular methods of volatile elicitation, wounding and freeze-thawing. Freeze-thawing distorted the volatile metabolism of both GLVs and PLVs relative to wounding, though this distortion differed between the two groups of volatiles. These results suggest that despite the structural similarity of these two volatile groups, they are differentially metabolized. Collectively, these results have allowed us to produce the most robust PLV pathway to date. To better elucidate the biological activity of PLVs, we show that PLVs induce maize resistance to the anthracnose pathogen, Colletotrichum graminicola, the effect opposite to that conferred by GLVs. Further analysis of PLV-treated and infected maize leaves revealed that PLV-mediated resistance is associated with early increases of oxylipin α- and γ-ketols, and later increases of oxylipin ketotrienes, hydroxytrienes, and trihydroxydienes. Ultimately, this study has produced the most up-to-date pathway for PLV synthesis, and reveals that PLVs can facilitate pathogen resistance through induction of select oxylipins.

Trichoderma virens colonization of maize roots triggers rapid accumulation of 12-oxophytodienoate and two ᵧ-ketols in leaves as priming agents of induced systemic resistance.

Two oxylipins 12-OPDA (12-Oxo-10(Z),15(Z)-phytodienoic acid) and an ᵧ-ketol, 9,10-KODA (10-oxo-9-hydroxy-12(Z), 15(Z)-octadecadienoic acid) were recently identified as important long-distance-induced systemic resistance (ISR) signals in Trichoderma virens-treated maize. On the other hand, jasmonic acid (JA), long believed to be a major signal of ISR, was not involved, as the JA-deficient mutant, opr7 opr8, retained the capacity for T. virens-triggered ISR. In order to further understand the biochemical basis for ISR priming in maize leaves, diverse oxylipins and phytohormones in the leaves of wild-type maize or ISR-deficient lox10-3 mutants treated with T. virens were quantified. This analysis revealed that 12-OPDA and two novel ᵧ-ketols, 9,12-KOMA (12-Oxo-9-hydroxy-10(E)-octadecenoic acid) and 9,12-KODA (12-Oxo-9-hydroxy-10(E),15(Z)-octadecadienoic acid), accumulated at high levels in ISR-positive plants. In support of the notion that 12-OPDA serves as a priming agent for ISR in addition to being a xylem-mobile signal, leaf pretreatment with this JA precursor resulted in increased resistance to Colletotrichum graminicola. Furthermore, the injection of 9,12-KODA or 9,12-KOMA in wild-type plants enhanced resistance against C. graminicola infection, suggesting that they play roles in ISR priming.

Relative contribution of LOX10, green leaf volatiles and JA to wound-induced local and systemic oxylipin and hormone signature in Zea mays (maize).

Green leaf volatiles (GLVs) and jasmonates (JAs) are the best-characterized groups of fatty acid-derived oxylipin signals that regulate wound-associated defenses. Beyond these two major groups of defense signals, plants produce an array of oxylipins in response to wounding, which possess potent signaling and/or insecticidal activities. In this study, we assessed the relative contribution of JAs and GLVs to wound-induced systemic signaling and the associated regulation of oxylipins in local and systemic tissues of maize (Zea mays). For this, we utilized GLV- and JA-deficient mutants, lox10 single and opr7opr8 double mutants, respectively, and profiled oxylipins in untreated leaves and roots, and in locally wounded and systemic leaves. In contrast to the studies in dicots, no systemic induction of JAs was observed in maize. Instead, a JA precursor, 12-OPDA, as well as ketols and C12/13 oxo-acids derived from 13-lipoxygenases (LOXs), were preferentially induced in both locally wounded and systemic unwounded leaves. Several 9-LOX-derived oxylipins (9-oxylipins) including hydroxides and ketones were also significantly induced locally. JA and JA-isoleucine (JA-Ile) were rapidly induced within 0.5 h, and were followed by a second increase in local tissue 4 h after wounding. GLV-deficient lox10 mutants displayed reduced levels of most 13-oxylipins, and elevated levels of several 9-oxylipins and the a-dioxygenase (DOX) product, 2-HOD. lox10 mutants were completely devoid of C6 volatiles and their C12 counterparts, and greatly decreased in C5 volatiles and their C13 oxo-acid counterparts. Thus, in addition to being the sole LOX isoform providing substrate for GLV synthesis, LOX10 is a major 13-LOX that provides substrate to several LOX branches that produce an array of 13-oxylipin products, including C5 volatiles. Interestingly, the rapid JA and JA-Ile increase at 0.5-2 h post-wounding was only moderately affected by the LOX10 mutation, while significantly reduced levels were observed at 4 h post-wounding. Combined with the previous findings that GLVs activate JA biosynthesis, these results suggest that both LOX10-derived substrates and/or GLVs are involved in the large second phase of JA synthesis proximal to the wound. Analyses of opr7opr8 mutants revealed that wound-induced oxylipin responses were positively regulated by JA signaling. The local and systemic accumulation of SA was not altered in the two mutants. Collectively, our results identified a subset of oxylipins strongly induced in wounded and systemic leaves, but their impact on insect defenses remain elusive. The lack of systemic induction of JAs points to substantial difference between systemic wound responses in studied dicots and maize. Our results show that GLV-deficiency and reduced JA in lox10 mutants had a greater impact on wound-induced local and systemic tissue oxylipin responses compared to the solely JA-deficient opr7opr8 double mutants. This suggests that GLVs or other LOX10-derived products heavily contribute to overall basal and wound-induced oxylipin responses. The specific roles of the GLV- and/or JA-dependent oxylipins in wound responses and defense remain to be further investigated by a combination of multiple orders of oxylipin-deficient mutants.

Green leaf volatiles and jasmonic acid enhance susceptibility to anthracnose diseases caused by Colletotrichum graminicola in maize.

Colletotrichum graminicola is a hemibiotrophic fungus that causes anthracnose leaf blight (ALB) and anthracnose stalk rot (ASR) in maize. Despite substantial economic losses caused by these diseases, the defence mechanisms against this pathogen remain poorly understood. Several hormones are suggested to aid in defence against C. graminicola, such as jasmonic acid (JA) and salicylic acid (SA), but supporting genetic evidence was not reported. Green leaf volatiles (GLVs) are a group of well-characterized volatiles that induce JA biosynthesis in maize and are known to function in defence against necrotrophic pathogens. Information regarding the role of GLVs and JA in interactions with (hemi)biotrophic pathogens remains limited. To functionally elucidate GLVs and JA in defence against a hemibiotrophic pathogen, we tested GLV- and JA-deficient mutants, lox10 and opr7 opr8, respectively, for resistance to ASR and ALB and profiled jasmonates and SA in their stalks and leaves throughout infection. Both mutants were resistant and generally displayed elevated levels of SA and low amounts of jasmonates, especially at early stages of infection. Pretreatment with GLVs restored susceptibility of lox10 mutants, but not opr7 opr8 mutants, which coincided with complete rescue of JA levels. Exogenous methyl jasmonate restored susceptibility in both mutants when applied before inoculation, whereas methyl salicylate did not induce further resistance in either of the mutants, but did induce mutant-like resistance in the wild type. Collectively, this study reveals that GLVs and JA contribute to maize susceptibility to C. graminicola due to suppression of SA-related defences.

Plant hairy roots enable high throughput identification of antimicrobials against Candidatus Liberibacter spp.

A major bottleneck in identifying therapies to control citrus greening and other devastating plant diseases caused by fastidious pathogens is our inability to culture the pathogens in defined media or axenic cultures. As such, conventional approaches for antimicrobial evaluation (genetic or chemical) rely on time-consuming, low-throughput and inherently variable whole-plant assays. Here, we report that plant hairy roots support the growth of fastidious pathogens like Candidatus Liberibacter spp., the presumptive causal agents of citrus greening, potato zebra chip and tomato vein greening diseases. Importantly, we leverage the microbial hairy roots for rapid, reproducible efficacy screening of multiple therapies. We identify six antimicrobial peptides, two plant immune regulators and eight chemicals which inhibit Candidatus Liberibacter spp. in plant tissues. The antimicrobials, either singly or in combination, can be used as near- and long-term therapies to control citrus greening, potato zebra chip and tomato vein greening diseases.