Early pest detection is the best insurance policy

Introduction

Powdery Mildew is arguable the most destructive Cannabis pest. It is an obligate biotroph that can vascularize into the plant tissue and remain invisible to a grower. It tends to emerge and sporulate 2 weeks into flowering thus destroying very mature crop with severe economic consequences. It is believed to travel in clones and its is not known if it travels in seeds. Other stressful events like cloning can trigger Powdery Mildew spore formation. While many fungicides are known to treat Powdery Mildew, many states are outlawing these fungicides due to cannabinoid extraction techniques enriching fungicides (Russo, Raber). Early detection and eradication may be the safest approach.

Background

MacPartland et al. reported L.taurica and P.macularis as the organisms responsible for powdery mildew on cannabis. Multiple unsuccessful attempts to amplify DNA sequences from these organisms led us to whole genome shotgun sequence the Massachusetts derived organism. This sequence assembly presented a novel organism not found in NCBI.

 

Powdery mildews (PM) are a unique fungi. They tend to have 180Mb genomes (40Mb is more common in fungi) and they are 90% repetitive Transposable elements. As a result these are a challenge to sequence and identify. Since plant domestication, they have become highly adapted to each host so it is rare to find PMs that cross species and some cereals even have more than 1 PM species (Blumeria) that specialize on it.

 

Under ideal conditions, some powdery mildews have a 4-7 day past innoculation (dpi) window where it remains invisible as it builds a network internal to the plant (dpi work performed in arabidopsis).  Research performed in a close cousin to cannabis, (Humulus lupulus  or hops) has demonstrated incubation periods up to 49 days with P.macularis. The work described below was performed to test if the Cannabis derived powdery mildew vascularized network is detectable with a youPCR DNA based test prior to conidiospore generation. Later stage powdery mildew infection and conidiospore generation results in rapid spreading of the fungus to other plants. This tends to emerge and sporulate 2 weeks into flowering thus destroying very mature crop with severe economic consequences. It is believed to travel in clones and it is not known if it travels in cannabis seeds, however evidence of P.macularis transmission in Hops seeds has been reported. Detection of this pest prior to sporulation can help eradicate powdery mildew before it infests a grow with hearty spores. DNA based tools could facilitate early detection and rapid removal of infected plant materials or screening of incoming clones.

 

Powdery Mildews often promote other synergistic endophytes after they infect and there are known antagonistic endophytes like Bacillus that some growers use for treatment. If you try to petri dish plate plants with PM, usually another penicillium or mold will grow instead. Without DNA sequencing, you might not know this as the colonies don’t sprout with a taxonomic I.D and visual identification can be misleading.

 

Another likely outcome with petri-dish plating is that you will get nothing to grow. Powdery mildew does not culture on common petri-dishes or 3M petri films which has made this a complicated topic for us. It is an obligate biotroph and there are no published papers on how to culture cannabis derived PM. Other PMs are cultured on living leaves.

 

DNA sequencing the PCR products derived from our PathogINDICAtor qPCR TYM test has been shown to pick up golovinomyces in the past albeit at very low read frequencies. After reviewing the DNA sequencing of the ITS regions whole genome shotgun assembly of the cannabis derived PM, we see 2 SNPs in our PathogINDICATor primers suggesting our qPCR TYM tests DO NOT detect Cannabis derived Powdery Mildew.

 

We consulted many industrial scale cultivators and cannabis testing labs regarding PM detection. Both parties emphasized that we will fail so much more crop if we pick up non-culturable PM and there will be no evidence for it on a petri-plate to back it up. Refactoring our TYM kits to detect PM could eliminate Medicinal Genomics from the microbial testing market by eroding the specificity for human pathogenic microbes provided with our qPCR safety products.

Specificity has always been our main selling point with qPCR and Aspergillus (also doesn’t grow well on petri-film) but there isn’t a single state that has PM on a list of dangerous pathogens like Aspergillus.

Nevertheless, we dug deeper as cultivators expressed a very strong interest in PM genetics. After sequencing the genome of Cannabis derived PM, we discovered its not Golovinomyces. Much of the draft genome is diverged (92% alignments with BLAST) and we will need a new +/- assay for this bug.

To this end we designed a colorimetric DNA amplification assay that can detect Powdery Mildew from a leaf punch prep on a $650 USB driven PCR device. This platform is known as youPCR and is excellent at Presence/Absence testing (+/-). The test cannot guarantee a quantitative assay until we can culture PM or get a pure source of its genome like we have with other ATCC controls. For surveillance and pest control a (+/-) assay is all that is required.


It remains an open debate as to the human health risks of Cannabis derived powdery mildew. While there are some anecdotal reports of allergies with PMs derived from other hosts, those allergies can’t be superimposed onto a different species. There are no states that require specific testing for these and no clinical literature supporting it as a threat to human health.

Case Studies

  1. Geographic Distribution. Powdery Mildew samples were collected from 4 locations in New England. Over 6 iterations of primer designs were used to optimize the sensitivity of the test and to reduce false positives. Results were followed up with sampling from several DNA isolations from California.

 

Expanded study on Powdery Mildew from CT, MA and NY

 

2. PCR detection vs Visual detection.-There is limited utility for a PCR test that can inform you of powdery mildews presence if you can already visibly see it.  The value of a DNA based test requires an understanding of the life cycle of PM. This fungi forms a mycelium network in the plant prior to sporulation (See WeBling et al. below). Sporulation is what spreads the disease throughout a grow. This incubation period can be 7-41 days in hops. Detection and elimination of infected plants prior to sporulation can prevent the spread of the disease. To address timing of detection sensitivity we tested various strains of PM that were believed to be resistant to PM, susceptible to PM, Visibly infected of PM and non-symptomatic susceptible to PM strains.

Sample Key (left to right)

  1. FSD-Nonsymptomatic) No visual signs of PM but near other infected plants
  2. FSD-Infected) Visual confirmation of PM. Known to be PM susceptible
  3. FSD-Infected#2) Visual confirmation of PM. Known to be PM susceptible
  4. GSC-NonSymptomatic) No visual signs but known to be PM susceptible
  5. GSC-Infected)  Visual signs of PM.
  6. BB-NonSymptomatic) PM Resistant strain.
  7. BB X 3- NonSymptomatic) Unknown cross of a PM resistant strain.
  8. BBR- NonSymptomatic) PM Resistant strain.
  9. BBR- NonSymptomatic#2) PM Resistant strain.
  10. BBRR-NonSymptomatic) Very PM Resistant strain.
  11. BBRR-NonSymptomatic#2) Very PM Resistant strain.
  12. Positive Control sample.
  13. Negative Control sample.

All resistant strains tested negative and all infected strains tested positive. A few NonSymptomatic strains (FSD & BBx3) had heterogeneous results likely related to sampling theory. At low mycelium density multiple 4mm hole punches may be required to for the earliest detection possible. Six 10 fold dilutions on a quantitative positive control imply we can detect single genomic copies of the species suggesting lack of assay sensitivity isn’t the cause for the heterogeneous results. However mycelium density below 2 square millimeters would result in sampling error.

To explore this we hyper sampled these strains.

2-3 Hole punches will provide earlier detection. We are exploring lysing 3 hole punches in one DNA prep to maximize sensitivity of detection while reducing youPCR costs.

Many infected strains were treated with lactobacillus and re-tested positive even though they did not visibly appear to be expressing spores. This implies the lactobacillus treatment may retard Conidia (spore) formation but does not eliminate the mycelium network.

Conclusions

Early detection of vascularized cannabis derived Powdery Mildew can be accomplished before sporulation and spreading of the disease. Early detection can drastically reduce fungicide usage and ensure safer grow environments and cleaner cannabis. Earliest detection may require triplicate sampling to provide best results.

icrs-2017-youpcr


Appendix Photos

Microscopic views of the original plant with Powdery Mildew-

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Next question-

Do other parts of the plant with no visual signs of Powdery Mildew have genetic evidence of its vascularization?

Sample 1pict1590pict1591

Positive for Powdery Mildew

 

Sample 2

pict1592pict1594

Negative for PM

Sample 3

pict1595pict1596

Positive for PM

 

Sample 4

pict1600pict1601Positive for PM

 

3/4 Hole punches from microscopically clean leafs from a plant that was visually positive with powdery mildew triggered a positive results.

screen-shot-2016-11-17-at-3-32-45-pm

 

These results confirm previous reports of powdery mildew being heavily vascularized even if not visible by eye. This presents an opportunity to use genetic tests on Clones of a Powdery Mildew tested Mother plant. While the test costs may be $15, a few tests on a mother can significantly reduce the likelihood that the 100 clones derived from that mother contain PM. While a single clone can cost $15, one may not need to test every clone from a mother.

A single cheap clone can destroy a whole grow.

screen-shot-2016-11-17-at-3-36-59-pm

WeBling et al. Stained for Powdery Mildew. One can see the mycelium network of PM inside the leaf even if powdery mildew is not visible to the human eye. DPI = Days Post Inoculation. HPI = Hours Past Inoculation.