First report of Pythium myriotylum causing root rot in Cannabis sativa (L.) in California.

In June of 2020 we observed greenhouse grown Cannabis sativa in Sonoma County, CA and Monterey County, CA showing stress symptoms: stunting, leaf chlorosis, and leaf senescence, when moved to flower production conditions. We uprooted symptomatic and healthy plants and observed disease symptoms only in symptomatic plants: reduced root mass, reduced root hair density, and necrosis. Roots and growth substrate samples were taken from infected and healthy plants for further analysis. Approximately one gram of soil was added to 20.0 mL deionized water and 5.0 mL of the resulting slurry was spread on water agar plates. Plates were rinsed free of soil after 24 hours of incubation, then incubated for an additional two days. Diffuse mycelial growth was observed on all soil plates from symptomatic plant pots and not healthy plant pots. Four subcultures were transferred to V8 media and grown for three days. Roots with brown lesions and healthy roots were surface sterilized by soaking in 0.1% sodium hypochlorite for five minutes, rinsed in sterile deionized water, and two-centimeter segments plated on V8 agar. After 24 hours mycelial growth was observed growing from the cut ends of the lesion roots and not the healthy-looking roots. Four subcultures were transferred to V8 and grown for three days. Mycelium from water sample isolates and root isolates were collected and DNA extractions performed using Quick DNA Fungi/Bacterial Kit (Zymo Research Irvine, CA, USA), then PCR amplified using ribosomal internal transcribed spacer (ITS) primers ITS100/ITS4 as described by Riit et al. and cytochrome oxidase I (COX) primers OomCox-Levup/OomCox-Levlo as described by Robideau et al. (Riit et al., 2016; Robideau et al., 2011). Amplicons from all eight isolates for each region were Sanger sequenced and the found to be identical, and consensus sequences deposited in Genebank with accession numbers MW436422 and MW448569 for ITS and COX sequences, respectively. Observation of cultures under light microscope revealed morphological characteristics congruent with P. myriotylum (Watanabe, 2002). Two V8 cultures isolated from roots were cut into approximately 2-3mm2 pieces and transferred to a one-liter flask of water. A negative control using clean V8 was also prepared. The flasks were placed on a rotary shaker and incubated at 150 rpm at ambient temperature for 48 hours. The resulting suspensions for zoospore and control treatments were observed under a light microscope and motile zoospores present in the water suspension from P. myriotylum cultures only. The zoospore suspension was then divided into six equal portions and applied to the soil of six C. sativa rooted cuttings in one-gallon pots. The control slurry was added to two C. sativa rooted cuttings. All plants were grown in a controlled environment for 28 days with 16-hour photoperiod. All plants were then removed from their pots and roots observed for symptoms. Plants that were treated with zoospore suspensions had tan to brown lesions on significant numbers of roots, and reduced root hair density compared to the plants treated with the control V8 agar suspension. Roots samples from all eight plants were then surfaced sterilized in bleach as previously described and five root sections from each plant plated on V8 media. After 48 hours mycelial growth was observed from root sections from P. myriotylum zoospore treated plants and not control plants. DNA extraction and PCR amplification for ITS and COX amplicons was performed from one representative culture for each plant, Sanger sequenced, and aligned with the previous sequences. All ITS and COX sequences were identical to the original sequences from greenhouse samples. P. myriotylum may cause root rot in C. sativa in greenhouse cultivation.

First report of Pythium ultimum causing crown rot in greenhouse grown Cannabis sativa (L.) in California.

In April of 2020 cuttings of Cannabis sativa (L.) in a greenhouse in San Mateo County, CA were observed collapsing, and further observation revealed: water-soaked stems, tan discoloration to the cortex, and discolored roots. The greenhouse irrigation system was supplied by a local stream. We collected one-liter water samples from: intake pond, reservoir tank, irrigation lines, and local potable water tap. Water samples were filtered and plated as described previously (Rollins et al., 2016). Filter papers were removed after 24 hours. Crown sections from four symptomatic plants and one asymptomatic plant were surfaced sterilized in 10% bleach for five minutes, rinsed in sterile deionized water, cut into four-millimeter long sections, and plated onto V8 media, then incubated at room temperature for three days. White mycelial growth was observed from foci within the print of the filter paper from all irrigation water samples but not the potable water supply sample. Similar mycelial growth was observed from plated crown tissue from symptomatic plants only. Observation under light microscope revealed characteristics congruent with P. ultimum, including aseptate hyphae and globose sporangia (Watanabe, 2002). Mycelia was collected for DNA extraction from each of the water and plant sample plates with DNA extractions performed using Quick DNA Fungi/Bacterial Kit (Zymo Research Irvine, CA, USA) and PCR amplified using primers ITS100/ITS4 as described by Riit et al. (2016). All amplicons were Sanger sequenced, aligned using SnapGene software (from GSL Biotech; available at snapgene.com), and compared to barcode referencPe sequences to identify the species using the BarCode of Life Database (BOLDsystems) within the National Center for Biotechnology Information nucleotide database. After trimming and aligning, all amplicons were found to be identical, yielding the 810-nucleotide long consensus ITS amplicon (accession MW114807), which aligned with Pythium ultimum ITS sequences (e.g., accession HQ643886.1) with 100% identity and homology. We then completed Koch's postulates by using pure cultures from root sections of P. ultimum to stem inoculate C. sativa plants. We used a three-millimeter corer to remove a disc of epidermis and applied a plug of pure culture to the wound. We inoculated 10 plants, with two plants mock-inoculated using clean V8 agar. Inoculation sites were wrapped in parafilm, and plants were grown in the greenhouse for 20 days. Stems of mock and oomycete inoculated plants were examined for callus formation and 30 centimeters of stem were excised from each plant. The mock inoculated plants had fully callused inoculation sites and were discolored only where wounded. P. ultimum inoculated plant inoculation sites were partially callused over and had tan discoloration of the cortex that extended 6.0 mm +/- 2.0 mm above and below the inoculation site. Stem segments above and below inoculation sites were surface sterilized and plated on V8 media as previously described and P. ultimum recovered from inoculated plants, confirmed as identical to the inoculum by ITS amplification and sequencing. Mock inoculated plant stem cultures yielded no oomycete growth. Together, these results indicate that P. ultimum has the ability to cause crown rot in C. sativa in greenhouse cultivation.

A phage display-selected peptide inhibitor of Agrobacterium vitis polygalacturonase.

Agrobacterium vitis, the causal agent of crown gall of grapevine, is a threat to viticulture worldwide. A major virulence factor of this pathogen is polygalacturonase, an enzyme that degrades pectin components of the xylem cell wall. A single gene encodes for the polygalacturonase gene. Disruption of the polygalacturonase gene results in a mutant that is less pathogenic and produces significantly fewer root lesions on grapevines. Thus, the identification of peptides or proteins that could inhibit the activity of polygalacturonase could be part of a strategy for the protection of plants against this pathogen. A phage-displayed combinatorial peptide library was used to isolate peptides with a high binding affinity to A. vitis polygalacturonase. These peptides showed sequence similarity to regions of Oryza sativa (EMS66324, Japonica) and Triticum urartu (NP_001054402, wild wheat) polygalacturonase-inhibiting proteins (PGIPs). Furthermore, these panning experiments identified a peptide, SVTIHHLGGGS, which was able to reduce A. vitis polygalacturonase activity by 35% in vitro. Truncation studies showed that the IHHL motif alone is sufficient to inhibit A. vitis polygalacturonase activity.

Insights into the Activity and Substrate Binding of Xylella fastidiosa Polygalacturonase by Modification of a Unique QMK Amino Acid Motif Using Protein Chimeras.

Polygalacturonases (EC 3.2.1.15) catalyze the random hydrolysis of 1, 4-alpha-D-galactosiduronic linkages in pectate and other galacturonans. Xylella fastidiosa possesses a single polygalacturonase gene, pglA (PD1485), and X. fastidiosa mutants deficient in the production of polygalacturonase are non-pathogenic and show a compromised ability to systemically infect grapevines. These results suggested that grapevines expressing sufficient amounts of an inhibitor of X. fastidiosa polygalacturonase might be protected from disease. Previous work in our laboratory and others have tried without success to produce soluble active X. fastidiosa polygalacturonase for use in inhibition assays. In this study, we created two enzymatically active X. fastidiosa / A. vitis polygalacturonase chimeras, AX1A and AX2A to explore the functionality of X. fastidiosa polygalacturonase in vitro. The AX1A chimera was constructed to specifically test if recombinant chimeric protein, produced in Escherichia coli, is soluble and if the X. fastidiosa polygalacturonase catalytic amino acids are able to hydrolyze polygalacturonic acid. The AX2A chimera was constructed to evaluate the ability of a unique QMK motif of X. fastidiosa polygalacturonase, most polygalacturonases have a R(I/L)K motif, to bind to and allow the hydrolysis of polygalacturonic acid. Furthermore, the AX2A chimera was also used to explore what effect modification of the QMK motif of X. fastidiosa polygalacturonase to a conserved RIK motif has on enzymatic activity. These experiments showed that both the AX1A and AX2A polygalacturonase chimeras were soluble and able to hydrolyze the polygalacturonic acid substrate. Additionally, the modification of the QMK motif to the conserved RIK motif eliminated hydrolytic activity, suggesting that the QMK motif is important for the activity of X. fastidiosa polygalacturonase. This result suggests X. fastidiosa polygalacturonase may preferentially hydrolyze a different pectic substrate or, alternatively, it has a different mechanism of substrate binding than other polygalacturonases characterized to date.

Localization and characterization of Xylella fastidiosa haemagglutinin adhesins.

Xylella fastidiosa is a gram-negative, xylem-inhabiting, plant-pathogenic bacterium responsible for several important diseases including Pierce's disease (PD) of grapevines. The bacteria form biofilms in grapevine xylem that contribute to the occlusion of the xylem vessels. X. fastidiosa haemagglutinin (HA) proteins are large afimbrial adhesins that have been shown to be crucial for biofilm formation. Little is known about the mechanism of X. fastidiosa HA-mediated cell-cell aggregation or the localization of the adhesins on the cell. We generated anti-HA antibodies and show that X. fastidiosa HAs are present in the outer membrane and secreted both as soluble proteins and in membrane vesicles. Furthermore, the HA pre-proteins are processed from the predicted molecular mass of 360 kDa to a mature 220 kDa protein. Based on this information, we are evaluating a novel form of potential resistance against PD by generating HA-expressing transgenic grapevines.

Xylella fastidiosa requires polygalacturonase for colonization and pathogenicity in Vitis vinifera grapevines.

Xylella fastidiosa is the causal agent of Pierce's disease of grape, an economically significant disease for the grape industry. X. fastidiosa systemically colonizes the xylem elements of grapevines and is able to breach the pit pore membranes separating xylem vessels by unknown mechanisms. We hypothesized that X. fastidiosa utilizes cell wall degrading enzymes to break down pit membranes, based on the presence of genes involved in plant cell wall degradation in the X. fastidiosa genome. These genes include several beta-1,4 endoglucanases, several xylanases, several xylosidases, and one polygalacturonase (PG). In this study, we demonstrated that the pglA gene encodes a functional PG. A mutant in pglA lost pathogenicity and was compromised in its ability to systemically colonize Vitis vinifera grapevines. The results indicate that PG is required for X. fastidiosa to successfully infect grapevines and is a critical virulence factor for X. fastidiosa pathogenesis in grapevine.

Persistence of Xylella fastidiosa in Riparian Hosts Near Northern California Vineyards.

The spread of Pierce's disease (PD) from riparian hosts to grapevines in California's northcoastal grape-growing region is a function of the proportion of Graphocephala atropunctata (blue-green sharpshooters [BGSSs]) that acquire Xylella fastidiosa from infected plant tissue. Riparian hosts that do not maintain sufficient X. fastidiosa populations for acquisition may not be significant inoculum reservoirs. We examined X. fastidiosa populations in systemically infected riparian hosts (California blackberry, California grapevine, elderberry, Himalayan blackberry, periwinkle) at two coastal locations (Mendocino and Napa) with two methods of quantitation (culturing and real-time polymerase chain reaction) from 2003 to 2004. In summer and autumn, X. fastidiosa populations were above the threshold for BGSS acquisition in periwinkle, Himalayan blackberry, and California grapevine at both locations. The only X. fastidiosa-positive plants detected in spring at both locations were periwinkle and Himalayan blackberry, suggesting that these species may contribute to long-term survival of X. fastidiosa. California blackberry and elderberry may not be important reservoirs of X. fastidiosa, given that very few plants of either species maintained infections. Higher X. fastidiosa populations in California grapevine, Himalayan blackberry, and periwinkle in Napa, relative to plants in Mendocino, may partially explain the higher PD incidence in Napa vineyards.