Fiber crops are an important group of economic plants Traditionally cultivated for fiber, fiber crops have also become sources of other materials such as food, animal feed, cosmetics and medicine Asia and America are the two main production areas of fiber crops in the world However, oomycete diseases have become an important factor limiting their yield and quality, causing devastating consequences for the production of fiber crops in many regions To effectively control oomycete pathogens and reduce their negative impacts on these crops, it is very important to have fast and accurate detection systems, especially in the early stages of infection With the rapid development of molecular biology, the diagnosis of plant pathogens has progressed from relying on traditional morphological features to the increasing use of molecular methods The objective of this paper was to review the current status of research on molecular diagnosis of oomycete pathogens on fiber crops Our search of PubMed identified nearly 30 species or subspecies of oomycetes on fiber crops, among which the top three species were Phytophthora boehmeriae, Phytophthora nicotianae and Pythium ultimum The gene regions that have been used for molecular identifications of these pathogens include the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene cluster, and genes coding for translation elongation factor 1α (EF-1α) and mitochondrial cytochrome c oxidase subunits I and II (Cox 1, Cox 2), etc We summarize the molecular assays that have been used to identify these pathogens and discuss potential areas of future development for fast, specific, and accurate diagnosis of oomycetes on fiber crops

https://www.mdpi.com/2223-7747/9/6/769/pdf

Molecular Diagnostics and Detection of Oomycetes on Fiber Crops.

Bast fibers and products derived from them are undergoing a resurgence in demand in the global market. However, fungal diseases have become an important factor limiting their yield and quality, causing devastating consequences for the production of bast fiber crops in many parts of the world. Thus, there is a high demand for effective control and prevention strategies against fungal pathogens. Having rapid, specific, sensitive, and cost-effective tests that can be used for early and accurate diagnosis of disease agents is an essential step of such strategies. The objective of this study was to review the current status of research on molecular diagnosis of fungal pathogens on bast fiber crops. Our search of PubMed identified nearly 20 genera of fungal pathogens on bast fiber crops, among which the five most common genera were Colletotrichum, Pythium, Verticillium, Fusarium, and Golovinomyces. The gene regions that have been used for molecular identifications of these fungi include internal transcribed spacer (ITS), translation elongation factor 1-α (EF-1α), s-tubulin, calmodulin (CAL), histone subunit 3 (H3), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), etc. We summarize the molecular assays that have been used to identify these fungi and discuss potential areas of future development for fast, specific, and accurate diagnosis of fungal pathogens on bast fiber crops.

https://www.mdpi.com/2076-0817/9/3/223/pdf

Molecular Diagnostics and Pathogenesis of Fungal Pathogens on Bast Fiber Crops.

The potential of species of Fusarium to cause significant economic losses in Cannabis sativa due to plant diseases and mycotoxin residues is the subject of this review. Sixteen species of Fusarium, reported as associated with cannabis production, are classified in six species complexes: Fusarium oxysporum, F. solani, F. incarnatum-equiseti, F. sambucinum, F. tricinctum, and F. fujikuroi. Taxonomy in this genus is the subject of debate, and removal of species in the F. solani Species Complex to the genus, Neocosmospora, has been proposed. Many species associated with C. sativa are also opportunistic pathogens of humans and animals. Species of Fusarium produce a myriad of mycotoxins, including at least three (deoxynivalenol, zearalenone, and fumonisin B) deemed the most important mycotoxins in human and animal foods. These chemicals vary from the very simple chemicals (moniliformin and butanolide) to the structurally complex depsipeptides (beauvericin and enniatin B) and trichothecenes (deoxynivalenol and its acetylated derivatives, diacetoxyscirpenol, and T-2-toxin). The robust strategies for disease management (e.g., exclusion of the pathogen, control of environment, and host resistance) are in development for this new crop and have primarily relied on management systems for other crops. Biopesticides have been labeled for use on C. sativa; however, few efficacy trials have been performed. Host resistance to these pathogens and transmission are also understudied. The new markets for C. sativa and its derivative products require knowledge for control of these important fungal diseases to provide safe products for human consumption.

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Diseases of Cannabis sativa Caused by Diverse Fusarium Species

Numerous Phytophthora and Pythium disease outbreaks have occurred in Europe following inadvertent introduction of contaminated ornamental plants. Detection and identification of pathogens are crucial to reduce risks and improve plant biosecurity in Europe and globally. Oomycete diversity present in roots and compost was determined in 99 hardy woody plants bought from nurseries, retailers and internet sellers, using both isolations and molecular analyses. Oomycete DNA was quantified using real-time PCR of environmental DNA from the plants using three loci: ITS, trnM-trnP-trnM and atp9-nad9. At least one oomycete species was isolated from 89.9% of plants using classical techniques. In total, 10 Phytophthora spp., 17 Pythium spp. and 5 Phytopythium spp. were isolated. Oomycetes were isolated from 86% of asymptomatic plants, but real-time PCR demonstrated that oomycetes were associated with all plants tested. More oomycete DNA occurred in composts in comparison with roots and filters from baiting water (a mean of 7.91 ng g−1, 6.55 × 10−1 ng g−1 and 5.62 × 10−1 ng g−1 of oomycete DNA detected in compost with ITS, trnM and atp9 probes, respectively); the ITS probe detected the highest quantities of oomycete DNA. No significant differences were found in quantities of oomycete DNA detected using real-time PCR in plants purchased online or from traditional retailers.

https://www.mdpi.com/2309-608X/7/2/87/pdf

Application of Real-time PCR for the Detection and Quantification of Oomycetes in Ornamental Nursery Stock

Lettuce microgreens are one of the most popular vegetables due to them being perceived as a “healthy food”, with high concentrations of nutrients, beneficial vitamins, and minerals. With a short vegetation period, they can be cultivated with minimum investment, and they are increasingly accepted by consumers, as they are healthy and easy to prepare. Lettuce has high ecological plasticity, but, despite this, its phenotypic expression, morphology, physiology, and anatomy are significantly influenced by environmental conditions. Lettuce microgreens contain higher quantities of phytonutrients and minerals and lower quantities of nitrates at the early stage of development than at the completely developed stage. The environmental conditions that influence the development of lettuce microgreens (and their quality) in a hydroponic system are as follows (average ideal values): light (400 W), photoperiodicity (12 h), light intensity (400 µmol m−2 s−1), colour spectrum (440-460 nm), temperature (20 ± 2 °C), and humidity (80 ± 5 %). The nutritional solution in a hydroponic system must be carefully monitored, by checking certain essential parameters such as the following (average ideal values): pH (6.3 ± 0.4), electrical conductivity (1.8 ± 0.2 mS), dissolved oxygen (6 mg L−1), and temperature (18 ± 2 °C). The analysis of expert literature reveals that there is a need to establish certain protocols for cultivating microgreens in hydroponic systems, to minimize the factors that can negatively influence the plants, in order to obtain higher concentrations of active substances.

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Influence of environmental and nutritional factors on the development of lettuce (Lactuca sativa L.) microgreens grown in a hydroponic system: A review

Efficacy of biofungicides against root rot and damping-off of microgreens caused by Pythium spp.

In September 2018, a hemp plant (Cannabis sativa L. cv. Dinamed) presented severe wilting, root rot, and mortality in a research greenhouse at the University of Connecticut in Storrs, CT. The hemp plant was grown in a 11.36-liter container with soilless peat-based potting mix (SunGro Fafard 3B, Agawam, MA, U.S.A.). The plants were in a polycarbonate greenhouse with a daytime heating set point of 20°C and a ventilation set point of 26°C under 18 h of supplemental lighting. Roots were collected from the symptomatic hemp plant, washed three times with sterile deionized water, blotted dry, and plated on PARP selective medium (Jeffers and Martin 1986). The plates were incubated in the dark at 21°C for 48 h. Mycelia resembling the morphology of Pythium species were observed in all plates. Following van der Plaats-Niterink’s key (van der Plaats-Niterink 1981), the isolate was identified as Pythium myriotylum based on the morphology of sexual and asexual structures. Colonies grown on sterile tall fescue leaves had filamentous, inflated, nonproliferating sporangia that ranged in width between 6 and 16 μm. Oogonia were 28 to 30 μm in diameter, globose, and had a smooth, thick wall (1 to 2 µm). The antheridia were curved, bulbous, and developed on branched stalks with multiple antheridia per oogonium. The appressoria were clavate, and oospores were mostly aplerotic. DNA was extracted from mycelial mats using the DNeasy Plant Mini Kit (Qiagen, Germantown, MD, U.S.A.). The cytochrome oxidase subunits I and II (coxI and coxII) (Martin 2000; Robideau et al. 2011) and the internal transcribed spacer (ITS) regions 1 and 4 (White et al. 1990) were amplified. The amplicons were sequenced and BLAST analyzed in NCBI GenBank. The isolate was confirmed to be P. myriotylum with ≥98% query coverage and 100% identity match to GenBank accession numbers LC440564.1, KJ595365.1, and KX671106. The nucleotide sequences obtained in this project have been assigned the GenBank accession numbers MN037880, MN037881, and MN022496 for coxI, coxII, and ITS, respectively. To confirm Koch’s postulates, three 68-day-old hemp plants (cv. Wife) were inoculated with P. myriotylum with 1 × 10⁵ oospores/ml obtained from three mycelial mats per pot. The mycelial mats, grown in V8 broth, were blended with 500 ml of deionized water, oospores were quantified using a hemocytometer, and the inoculum was applied directly in the potting medium so that no leaching occurred. The hemp plants were grown in 11.36-liter containers in a growth chamber set at 24°C with 65% humidity under 16 h of light. After 20 days, two of the three inoculated plants were wilted, and the control plants were symptomless. Necrotic roots were collected from all the plants. Ten root fragments were plated on PARP selective medium (replicated four times, one plate per replicate). The mycelial growth emanating from roots resembled Pythium on isolation plates from the symptomatic plants, and no growth was observed from roots of noninoculated plants or the asymptomatic plant. Colonies isolated from the root tissue morphologically resembled the originally isolated organism, thus fulfilling Koch’s postulates. To our knowledge, this is the first report of P. myriotylum being detected on a hemp plant in the United States.

First Report of Root Rot and Wilt Caused by Pythium myriotylum on Hemp (Cannabis sativa) in the United States

In August 2016, lettuce (Lactuca sativa L.) plants presented root rot, severe wilting, and high incidence of mortality in New Haven County, CT. The plants were grown in a hydroponic deep-water culture system in a commercial greenhouse. Mature plants were stunted, the lower leaves were chlorotic, the roots were necrotic, and some plants collapsed. Lettuce roots were collected from several symptomatic plants, washed three times with sterile deionized water, blotted dry, and plated on PARP-V8 selective medium (0.005 g of pimarcin, 0.25 mg of ampicillin, 0.01 g of rifampicin, 0.10 g of pentachloronitrobenzene, 200 ml of clarified V8 juice, and 800 ml of distilled water) (Moorman et al. 2002). The plates were incubated in the dark at 21°C. Mycelium resembling the morphology of Pythium species was observed in all plates after 3 days. The isolate was identified as Pythium dissotocum based on the morphology of sexual and asexual structures following van der Plaats-Niterink’s key (van der Plaats-Niterink 1981). Colonies grown on V8-juice agar had filamentous sporangia with a dendroid structure, the oogonia were 23 μm in diameter and subglobose, the antheridia developed on unbranched stalks and were sessile, and the oospores were aplerotic. DNA was extracted from mycelial mats using a DNeasy Plant Mini Kit (Qiagen, U.S.A.), and the internal transcribed spacer (ITS) region was amplified using universal primers ITS1 and ITS4 (White et al. 1990). BLAST nucleotide analyses against publicly available Pythium sequences in GenBank confirmed that the isolates obtained were P. dissotocum with 97% query coverage and 99% identity match to GenBank accession number KM061701.1. The nucleotide sequence has been assigned the GenBank accession number MG993546. To confirm Koch’s postulates, 14-day-old lettuce seedlings, cultivar Rex (n = 25), were inoculated with 1 × 10⁵ zoospores of the isolate of P. dissotocum produced on V8 broth. After 7 days, symptoms appeared as reduced growth, necrotic roots, and chlorotic leaves. Control plants were symptomless and healthy. Roots were collected from each infected lettuce plant, and four root fragments were plated on PARP-V8 selective medium. Mycelial growth resembling Pythium was observed in all plates from inoculated plants, and no growth was observed from roots of noninoculated plants (n = 25, 1 plate per plant). The putative P. dissotocum was reisolated from the plates; DNA was extracted from mycelial mats using the same methods described above and was confirmed to be Pythium using primers ITS1 and ITS4. The amplicon was the same size as for the P. dissotocum recovered from the original plants. This is the first report of P. dissotocum being detected on lettuce in Connecticut. P. dissotocum reduces crop yield and was first reported in 1986 as a root pathogen on hydroponic lettuce in Arizona (Stanghellini and Kronland 1986). There are currently no registered synthetic-chemical fungicides in the United States for the suppression of Pythium species in hydroponic lettuce (Utkhede et al. 2009). Prevention of this disease is recommended by controlling temperature and salt levels in the nutrient solution, sanitizing surfaces, and applying preventative biological fungicides registered for Pythium root rot.

First Report of Pythium dissotocum Causing Pythium Root Rot on Hydroponically Grown Lettuce in Connecticut

Oomycetes and fungi were recovered from coconut coir and rockwool substrates where marijuana (Cannabis sativa L. cv. Silver and Citron) plants with root rot and wilt symptoms were grown in a commercial growing facility in Connecticut. The objectives of this study were to identify the isolates collected from these substrates, determine the pathogenicity of the isolates on hemp seedlings in vitro and in vivo, and evaluate the pathogens’ sensitivity to mefenoxam. Pythium and Globisporangium isolates were identified by sequencing the mitochondrially-encoded cytochrome oxidase genes (COI and COII) and Fusarium sp. with the translation elongation factor (EF-1α) region and internal transcribed spacer region (ITS4 and ITS5) genes. Three isolates were identified as Globisporangium irregulare (formerly Pythium irregulare), 21 isolates were Pythium myriotylum, and one was Fusarium oxysporum. All the isolates tested were pathogenic to hemp plants in vitro and in vivo, with disease incidence between 6.7 and 100%. Inoculated plants were smaller by 32% or more compared with the non-inoculated control. On average, hemp plants infected with Pythium myriotylum produced the lowest biomass and relative greenness values. None of the Pythium and Globisporangium isolates were resistant to mefenoxam—all were sensitive to ≥ 5 µg·mL-1 mefenoxam. This is the first report of G. irregulare causing root rot on marijuana and hemp plants. The results of this study provide information about the characteristics of pathogens that can be found potentially in soilless substrates in controlled environment agriculture.

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Pathogenicity and Mefenoxam Sensitivity of Pythium, Globisporangium, and Fusarium Isolates From Coconut Coir and Rockwool in Marijuana (Cannabis sativa L.) Production

Chlorine is a disinfectant commonly used to treat water. The United States Environmental Protection Agency (USEPA) has set a standard limit of up to 4 mg·L−1 chlorine for drinking water. The objective of this project was to identify chlorine phytotoxicity thresholds on ‘Rex’ lettuce (Lactuca sativa) when the water source contained chlorine levels within the USEPA standard limits. The nutrient solution to grow lettuce was prepared with reverse osmosis–treated water treated with 0, 0.5, 1, 1.5, 2, and 4 mg·L−1 chlorine and then fertilizers were added. Lettuce plants were grown in a deep-water culture hydroponic system. Visual toxicity symptoms on leaves, relative leaf greenness, and fresh and dry biomass were measured. Our results indicate that irrigation water sources with ≥1 mg·L−1 chlorine used to prepare nutrient solutions can cause phytotoxicity in lettuce plants in just 3 days. Compared with the untreated control, lettuce shoot biomass was lower by 30%, 55%, 66%, 83%, and 92% at 0.5, 1, 1.5, 2, and 4 mg·L−1 of chlorine, respectively. Water sources with ≥ 1 mg·L−1 chlorine can cause significant marketable yield reduction in lettuce grown in deep-water culture. 

Cora S. McGehee

Papers

Efficacy of biofungicides against root rot and damping-off of microgreens caused by Pythium spp.

First Report of Root Rot and Wilt Caused by Pythium myriotylum on Hemp (Cannabis sativa) in the United States

First Report of Pythium dissotocum Causing Pythium Root Rot on Hydroponically Grown Lettuce in Connecticut

Pathogenicity and Mefenoxam Sensitivity of Pythium, Globisporangium, and Fusarium Isolates From Coconut Coir and Rockwool in Marijuana (Cannabis sativa L.) Production

Irrigation Sources with Chlorine Drinking Water Standard Limits Cause Phytotoxicity on ‘Rex’ Lettuce Grown in Hydroponic Systems