To be Host or not to be: Finding Sustainable Management through Endophyte Interactions

1.- Development of molecular detection protocols for V. dahliae lineages 4A and 4B.

The first objective of this project is to develop VCG/lineage-specific markers for molecular detection of V. dahliae 4A and 4B.

Verticillium dahliae isolates

Four isolates of V. dahliae representative of the lineages 4A and 4B were selected for whole genome sequencing. T003 and S-228 belong to V. dahliae lineage 4A and U073 and S-39 to lineage 4B. These individuals were recovered in potato fields from Pennsylvania and Ohio and were isolated from a naturally infected potato plant (T003), soil from potato fields (S-228 and S-39), and an asymptomatically-infected rotational oat cultivated in a potato field (U073) (Table 3). Pathogenicity of the isolates has been confirmed in previous works. These isolates are currently maintained in soil vials at the V. dahliae culture collection in Jimenez-Gasco’s lab, The Pennsylvania State University, University Park, PA.

Whole genome sequencing and assembly

Fungal mycelia were grown on cellophane film on petri plates filled with potato dextrose agar (PDA, Difco™, Franklin Lakes, NJ). Mycelia was harvested and freeze-dried, and around 5 grams of lyophilized tissue from each isolate were sent to Beijing Genomics Institute (BGI, Shenzhen, China) for whole genome sequencing.

Isolates T003 and U073 genomes were sequenced using a de-novo hybrid whole genome sequencing approach which involves a combination of PacBio long reads (Pacific Biosciences technology, Menlo Park, CA, US) and short paired-end reads using Illumina HiSeq 4000. PacBio long reads were trimmed, quality-filtered and assembled into scaffolds using Canu 1.7.1 (Koren et al. 2017). Illumina short reads were trimmed and quality-filtered using Trimmomatic 0.38 (Bolger et al. 2014). Trimmed short reads were aligned to the scaffold draft assembly with BWA 0.7.17 (Li 2013) mem option and default parameters for paired-end reads. Nucleotide errors in the draft scaffold assembly were corrected with the aligned short reads using Pilon 1.23 (Walker et al. 2014). Isolates S-39 and S-228 genomes were sequenced using only Illumina HiSeq 4000. Illumina short reads were trimmed and quality-filtered using Trimmomatic 0.38 and de-novo assembled into scaffolds using SPAdes 3.13 (Bankevich et al. 2012). Qualities of the assemblies were checked with Quast 5.0.2 (Mikheenko et al. 2018). Completeness of the genome assembly in terms of gene content was analyzed using de-novo gene prediction with GlimmerHMM (Majoros et al. 2004), and single-copy orthologue content with BUSCO using fungus and conserved-genes-finding options (Simão et al. 2015).

Development of V. dahliae lineage 4A and 4B molecular markers

Lineage-specific (LS) genomic regions for V. dahliae lineage 4A and 4B were identified by selecting genomic nucleotide sequences from isolates of lineage 4A (T003 and S-228) and lineage 4B (U073 and S-39) that were present only in the genome assemblies of their respective lineage and not in other V. dahliae lineages reference genome assemblies.

First, Illumina short reads from T003 and U073 were aligned to their respective genomes assemblies using BWA with mem option. Only mapped reads were then aligned to genome assemblies of V. dahliae lineage representatives 1A/B (Gwydir1A3 and Vd991), 2B⁸²⁴ (VdLs.17 and JR2), 2A (12161 and 12158), 4A (S-228) and 4B (S-39) using the same alignment algorithm (Table 2). Once lineage-specific reads were identified, these were de-novo assembled into longer fragments (i.e. scaffolds) using Spades with default options. Lineage-specific scaffolds were analyzed for: i) potential number of copies in their reference genomes by aligning them back using BWA mem; ii) presence of predicted genes or coding sequences (CDS) using GlimmerHMM in Quast; iii) presence of homologous gene sequences using a gene-annotated genome from Verticillium longisporum available as reference in WebAUGUSTUS prediction tool (Hoff and Stanke 2013); and (iv) presence of repetitive elements with the RepeatMasker Web Server selecting RMBlast as search engine, part of the NCBI Blast tool suite, and fungi DNA option (Smit 2013). For the design of PCR-based LS markers, we selected genomic nucleotide sequences that had the following conditions: i) they only aligned to one position in the genome assemblies of their respective lineage, and did not have insertions, deletions or significant nucleotide mismatches; ii) they have a CDS and was not homologous to CDSs from V. longisporum; and iii) did not have repetitive elements.

Second, PCR-based LS markers were designed using Primer-BLAST web tool selecting a range of 80-150 bp for PCR product size, and a 57º C minimum, 60º C optimum and 63º C maximum primer melting temperatures. Organism specificity of the primers was ensured using the nucleotide reference database and delimiting the organisms to bacteria, fungi and oomycete. For each LS coding genomic sequence, we selected the primer pairs with the best characteristics for PCR and Quantitative-PCR (Q-PCR) reactions, which are: i) primer sizes with 18-22 nucleotides; ii) GC nucleotide content between 50-60%; iii) having a G or C nucleotide in the last 5 bases of the primer; iv) not having repeats of 4 or more adjacent identical nucleotides in the primer sequence; and v) the lowest scores for self-complementarity and self 3’-complementarity. Annealing temperatures (T_a) of the primers were estimated using the New England Biolabs Tm Calculator (New England Biolabs, Ipswich, MA) and some primer pairs were initially tested on DNA samples from V. dahliae lineages 4A and 4B using the estimated T_a (Table 1 and 2). The conditions of the PCR reaction for the selected pairs of primers were optimized using a gradient of annealing temperatures ranging from 54º C to 64º C and DNA samples from T003 isolate (V. dahliae lineage 4A) and U073 isolate (V. dahliae lineage 4B) respectively. PCR reaction mixtures (25 μl) consisted of 5-10 ng of fungal DNA template, 400 nM of each primer and 12.5 μl OneTaq® 2X Master Mix with Standard Buffer (New England BioLabs, Ipswich, MA). Conditions of the PCR reactions included an initial denaturation at 96°C for 4 min, followed by 35 cycles of denaturation at 96°C for 1 min, annealing at corresponding temperature for 30 s, elongation at 72°C for 1 min, and a final elongation step at 72°C for 6 min.

Primer pairs were tested on a range of DNAs obtained from representatives of the different evolutionary clades from V. dahliae lineages 4A and 4B using the optimized PCR conditions of each marker (Table 1). In addition, primers were also tested on DNA from V. dahliae representatives of lineages 1A/B, 2A, 2B⁸²⁴, 2B³³⁴, 3 and 6 to analyze their lineage-specificity amplifications (Table 3).

Quantification of V. dahliae lineages 4A and 4B in environmental samples

A protocol to quantify the amount of V. dahliae lineage 4A and 4B DNAs present in samples was developed at Wright Lab (Huntingdon, PA) using the PCR-based markers designed in this study. Digital Droplet PCR (ddPCR) is a precise method to quantify absolute amounts of nucleic acids without a standard curve and without dependence on amplification efficiency. The ddPCR is a relatively new technology but provides fast and more robust detection and quantification methods than previous technologies such as Real-Time Quantitative-PCR. The ddPCR protocol for lineage 4A and 4B markers was tested on DNA extracted from field soil samples infested with T003 isolate (V. dahliae lineage 4A) and U073 isolate (V. dahliae lineage 4B). Those field soil samples were collected from our research microplots at the Russell E. Larson Agricultural Research Center, The Pennsylvania State University. As part of a separate on-going experiment, the research microplots soil was infested with different V. dahliae treatments: T003 isolate (V. dahliae lineage 4A), U073 isolate (V. dahliae lineage 4B) and mixes at different ratios of both isolates (50:50, 20:80, and 80:20). Each set of microplots were cultivated with a monoculture of potato 'Snowden' (highly susceptible to Verticillium wilt), a monoculture of oat 'Armor' (an asymptomatic host) and a rotation of both.

Briefly, DNA from soil samples was extracted using ZymoBiomics DNA/RNA mini prep (Zymo Research) following manufacturer’s instructions. Samples without soil input were also extracted to serve as extraction controls (negative controls for ddPCR). DNA sample extracts were then quantified with Qubit 1X dsDNA high sensitivity assay on a Qubit 4 (Thermo Fisher) before being stored at -20 C until ddPCR set up. During set up, samples were normalized into a 96 well plate for ddPCR to a mass of 25 ng of DNA per sample and a master mix containing respective primer pairs (sequences below) of forward and reverse primers (final concentration: 0.25 µM forward, 0.1 µM reverse) were combined with EvaGreen Supermix (Biorad) was made for each primer set. The master mix was then added to the normalized sample wells and adjusted with nuclease free water to a final volume of 22 µL. Negative controls from extraction had 11 µL of extraction blank eluant added and no template controls of RT-grade PCR water were included following samples. Droplets were then generated on an Automated Droplet Generator (BioRad) and underwent PCR following EvaGreen reaction conditions as follows: Enzyme activation at 95 C for 5 minutes; followed by 40 cycles of 95 C for 30 s, then annealing and extension at 60 C for 60 s; followed by a 4 C hold for 5 minutes, then signal stabilization at 90 C for 5 minutes, before holding at 4 C until droplet reading. Once PCR was complete, droplets were read on a QX200 Droplet reader and resulting copies/µL and copies/20 µL reaction generated via QuantaSoft (Biorad).

2. Identify which crops species used in rotation with potato are asymptomatically infected with V. dahliae and more specifically, which pathogen VCGs/lineages (4A, 4B) infect them.

The second objective of this project is to detect V. dahliae lineages infecting plant species cultivated under rotation in potato fields, and moreover, to identify which plant species get infected by which lineage/VCG. This is quite important since different lineages/VCGs show different aggressiveness to different plant hosts and therefore, they pose different threats to the sustainability of the disease management strategy. V. dahliae-infected plant samples have been collected through Robert Leiby, PA Co-Operative Potato Growers, and Dr. Sara May, director of the Penn State Plant Disease Clinic. Samples included different cultivars of potato, pepper and watermelon grown in commercial production fields in PA. Plant samples were washed with tap water. Stem tissues were finely sliced and lyophilized for further DNA extraction. Currently, more than 25 plant samples have been collected from about 5 farmers from different locations in PA. During summer 2018, weather conditions in PA were not conducive for Verticillium wilt and there were no reported symptomatic thus limiting our sample collection. Therefore, that limited our sample collection in that season. DNA of the plant samples will be extracted using DNeasy Plant mini kit (Qiagen, Hilden, Germany).

Literature cited

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Workshops and training performed

Since 2017, I have taken several workshops to train myself in the type of analysis I need to perform in my objectives. I plan to share my knowledge with other students through workshops so they can apply as well in their research:

• Whole genome sequence analysis workshop by Jasna Kovac. The Microbiome Center, The Pennsylvania State University, University Park, PA. November 2, 2018.
• Launching the Agricultural Microbiomes Research Coordination Network. Agricultural Microbiomes Research Coordination Network. International Congress for Plant Pathology (ICPP) 2018. Boston, MA. July 28, 2018.
• Biological Data Analysis: The Right Way, The Second Boot Camp on Data Reproducibility by NIH funded Computation, Bioinformatics and Statistics (CBIOS) Predoctoral Training Program. The Pennsylvania State University, University Park, PA. July 10-14, 2017.
• Genome Assembly workshop by Qi Sun. Institute of Biotechnology, Cornell University, Ithaca, NY. May 24-31, 2017.