REGALIA® BIOPROTECTANT

REGALIA® BIOPROTECTANT IN PLANT DISEASE MANAGEMENT

Hai Su, Russell Blair, Tim Johnson and Pamela Marrone, Marrone Bio Innovations, Inc., Davis, California, 95618,
USA, describe the modes of action and biological efficacy of this new biological fungicide/bactericide

Keywords: biopesticide, disease, fungicide resistance, induced resistance, ISR,

bacterial diseases, and mancozeb and mefenoxam in control-
knotweed, plant extract, Regalia, Reynoutria sachalinensis, REYSA, SAR ling downy mildew. To enhance soilborne disease control and
increase emergence, multiple delivery methods can be used,
such as seed treatment, soil drenches, irrigation applications,
and dipping seedlings prior to transplanting.
Regalia® as a biofungicide The successful commercialization of Regalia® is the result
Plants have long been used as herbal medicines for treatment of extensive research into efficacy and formulations, demon-
of human diseases. However, only a limited number of plant stration of all application options for efficacy, and innovative
extracts have been developed into fungicides for plant disease marketing strategies. Integration of plant extracts in disease
control. The commercialization of Regalia® illustrates the management programs can increase yields and quality, reduce
potential for plant extracts to be an important tool for disease fungicide resistance in pathogens, lower residue of synthetic
management in both organic and conventional production fungicides in plants and soil, and protect human health and
systems and emphasises the need for additional plant extracts the environment.
to be explored as a source of biopesticides.
Regalia® is formulated from the extract of giant knotweed
(Reynoutria sachalinensis (REYSA)) and is now becoming
widely used in commercial crop production. Giant knotweed Mode of action of Regalia®
is used as a food in many countries, especially Asia. Knotweed Reynoutria sachalinensis (Figure 1) (Syn. Polygonum sacha-
was formerly formulated as Milsana® in the1980s and was linensis, Fallopia sachalinensis) belongs to the plant family
tested in field trials through the 1990s. Trial demonstrations Polygonaceae and contains anthraquinones such as resvera-
were limited to cucumber powdery mildew (Sphaerotheca trol, physicon and emodin, which are known to be antimi-
fuliginea) and a few other diseases such as Botrytis fruit rot crobial. The current formulations available are Regalia® SC
(Botrytis cinerea) and wheat powdery mildew (Blumeria (5% w/w), Regalia® Bioprotectant Concentrate (5% w/w),
graminis f. sp. tritici). Since the 2009 introduction as the and Regalia® Maxx (20% w/w), all of which have better effi-
reformulated product Regalia® by Marrone Bio Innovations, cacy and broader spectrum than the earlier Milsana® product
Inc., extensive tests have been conducted in the laboratory, and also provide new tools for both conventional and organic
glasshouse and field on multiple crop-disease systems to eval- growers (Su et al. 2009). Extensive research results show that
uate its efficacy for disease control. Test results demonstrate the extract of R. sachalinensis can induce plant resistance by:
the efficacy of Regalia® applied as a foliar spray in controlling Induction of phytoalexins and phenolic compound production;
a wide range of fungal and bacterial diseases, such as powdery Increase in production of defense-related proteins;
mildew of cucurbits, downy mildew of lettuce (Bremia lactu-
cae), Botrytis of grapes and strawberries, bacterial spot of • Introduced into North America
as ornamental and for livestock
tomatoes and peppers (Xanthomonas campestris pv. vesica- fodder
• Widely distributed invasive,
toria), Cercospora on soybeans (Cercospora kikuchii) and riparian weed
• Insects and birds feed on Reysa
bacterial canker on citrus (Xanthomonas axonopodis pv. • Livestock foraging a control
citri), amongst others. method
• In Buckwheat family, a common
The broad spectrum of disease control by Regalia® relies food in Asian cultures
• Celery and rhubarb close
on the unique mechanism of induced plant resistance. Studies relatives
in plants show that Regalia® treatment increases the activity • Hybridizes with Japanese
knotweed
of chalcone synthase and chalcone isomerase in the phenyl-
propanoid pathway and induces the production and accumu-
lation of phytoalexins. Simple phenolic compounds, which
are fungitoxic, also accumulate. Additional studies show that
Regalia® increases the papillae formation at pathogen pene-
tration sites as well as the liginification of plant cell walls.
Activities of pathogenesis-related protein (PR-proteins) such
as chitinase, glucanase, and peroxidase are also increased.
Regalia® is an excellent tool for fungicide resistance
management and is synergistic with commonly used fungi-
cides, such as azoles, strobilurins, and sulfur in controlling
powdery mildew and leaf spot diseases, copper in controlling Figure 1. Reynoutria sachalinensis.

3 0    O u t l o o k s o n Pe s t M a n a g e m e n t – F e b r u a r y 2 0 1 2 DOI: 10.1564/23feb09
© 2012 Research Information Ltd. All rights reserved. www.pestoutlook.com
 REGALIA® BIOPROTECTANT
Accumulation of reactive oxygen species; reactive oxygen species (ROS) such as superoxide radicals
Lignification and papilla formation in cell walls. (∙O2-), hydrogen peroxide (H2O2), and hydroxyl radicals
(∙OH). These compounds are released from plant cells to
cause cell death in order to restrain pathogen growth resulting
Induction of phytoalexin production
in a hypersensitive reaction (HR). ROS also serve as defense
Daayf et al. (1997) first demonstrated that elevated phyto- signaling for induced resistance-related pathways (Bolwell &
alexin and phenolic compounds measured after REYSA Wojtaszek, 1997). Thus, ROS are often used as markers for
treatment are resposible for the strong resistance of cucum- measuring plant resistance to diseases.
ber to powdery mildew (S. fuliginea). Wurms et al. (1999) Randoux et al. (2006) found increased accumulation of
also found that increased levels of phenolic compounds reactive oxygen species (ROS) such as H2O2 in wheat after
after REYSA treatment increased the resistance of wheat to treatment with REYSA and increased resistance to powdery
powdery mildew (Blumeria graminis f.sp. triciti). mildew. Additionally, Věchet et al. (2005) also treated wheat
Fofana et al. (2002) discovered increased levels of mRNA with REYSA and after inoculation with powdery mildew
and activity of chalcone synthase (CHS) and chalcone isome- (Blumeria graminis f.sp. triciti) they obtained good disease
rase (CHI), and elevated levels of flavonoid compounds due control in REYSA treated plants.
to REYSA treatment, which lead to a high level of resistance
in cucumber to powdery mildew. By interrupting the flavo-
noid pathway in cucumber plants, Fofana et al. (2005) could Wall apposition
down-regulate chalcone synthase in the flavonoid pathway Accumulation of lignin on cell walls helps plants fight against
and reduce resistance to powdery mildew. Similarly, McNally plant pathogen invasion, and hence is considered another
et al. (2003) further detailed and confirmed that C-glycosyl expression of induced resistance (Vance et al., 1980). For
flavonoid phytoalexin production after REYSA treatment example, Hammerschimdt and Kuć (1982) found that lignifi-
increased resistance in cucumber to powdery mildew. cation is responsible for resistance to different pathogens such
Daayf et al. (2000) reported that the phenolic compounds as Colletotrichum lagenarium, and Cladosporium cucumer-
ρ-coumaric acid, caffeic acid, ferulic acid and ρ-coumaric inum in cucumber. Wurms et al. (1999) reported increased
acid methyl ester accumulated after REYSA treatment. These lignification and thickening of cell walls in REYSA-treated
compounds increased resistance in cucumber to S. fuliginea plants. Papillae formed at the penetration sites of the powdery
in vivo and showed fungitoxic effects on Botrytis cinerea, mildew fungus (S. fuliginea) to prevent penetration of germ
Pythium ultimum and P. aphanidermatum. tube of the pathogen. Formation of papillae in REYSA-treated
Additional evidence by Zavareh et al. (2007) showed that plants was also confirmed by Fofana et al. (2005).
the activity of phenylalanine ammonia-lyase (PAL) in REYSA
extract-treated cucumber increased rapidly and resulted in
resistance to powdery mildew. PAL is a critical enzyme in the Additional evidence
flavonoid pathway and it is typically used as an indicator of Some of the active ingredients such as physcion and emodin
resistance to stress, such as disease, drought, and flood. in REYSA and other plant extracts induce resistance in grape-
vine
Pathogenesis-related (PR) proteins It has been shown that REYSA controls powdery mildew
(Uncinula necator) on grapevine (Bervejillo et al., 1999).
Zavareh et al. (2007) studied the response in cucumber Schnee et al. (2008) showed that stilbenic phytoalexin
inoculated and non-inoculated with S. fuliginea after treat- accumulation is modulating the resistance in grapevine to
ment with REYSA. They found that the activity of peroxi- powdery mildew. Godard et al. (2009) used Rheum palmatum
dase, a plant defense-related protein (van Loon et al., 2006), (Rhubarb, family Polygonaceae) and Frangula alnus (Alder
increased significantly in similar patterns in treated tissues of Buckthorn, family Rhamnaceae), which contain emodin,
pathogen-inoculated and non-inoculated plants. physcion, and other related (anthraquinone-rich) compounds,
Schneider and Ullrich (1994) investigated the mechanism of to treat grape vines. These plant extracts induced stilbenic
REYSA by studying powdery mildew (S. fuliginea) on cucum- phytoalexin production, increased activity of peroxidase,
ber and bacterial speck (Pseudomonas syringae pv. tobaci and induced the hypersensitive reaction (HR), and inhibited spore
P. syringae pv. pisi) on tobacco in parallel. Both cucumber germination of Plasmopara viticola (vine downy mildew).
and tobacco plants treated by REYSA had increased activities The control of plant pathogens through induced resistance
of chitinase, beta-1,3-glucanase (PR-proteins) and PAL, and is universal among various plant and plant pathogen species
resulted in significantly lower disease severity compared with (Bostock, 2005; Feys & Parker, 2000). REYSA induces
the untreated control. systemic resistance and works through multiple simultane-
Increase of PR proteins and of resistance to powdery ous mechanisms in the cellular level, most important of which
mildew (Blumeria graminis) was also detected in wheat after lead to increase in phytoalexins, phenolics, PR-proteins, reac-
treatment with REYSA (Véchet et al., 2005). tive oxygen species, and cell wall lignification.

Production of reactive oxygen species (ROS) Synergy

Among all plant species, a common response to pathogen Combination (tank mix) of Regalia® and other commercial
infection or physical damage is to generate elevated level of fungicides can be an effective and efficient measure in increasing

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 REGALIA® BIOPROTECTANT
pathogen control efficacy and managing fungicide resistance these results found in the greenhouse tests. The examples of
in pathogens. Greenhouse and growth chamber experiments synergy with azoxystrobin and myclobutanil in a greenhouse
were conducted to evaluate the efficacy of Regalia® in combi- trial are shown in Figure 2 and 3.
nation with commonly used chemical and biological fungi-
cides in controlling powdery mildew on cucurbits and lettuce
downy mildew. The results show a statistically significant Seed treatment uses
synergistic effect with Regalia® in a tank mix with azoxys- Regalia® can also be applied as a seed coating or drenched
trobin (Quadris®), myclobutanil (Rally® 40W), quinoxyfen in soil to control soilborne diseases caused by Rhizoctonia
(Quintec®), or triflumizole (Procure®), and other commercial solani or Pythium ultimum in soybean and cotton. In our
fungicides in controlling powdery mildew on cucumber in studies, Regalia® alone, or mixed with azoxystrobin (Quad-
repeated tests. Only an additive effect with no synergy was ris®), fludioxonil (Scholar®), or mefenoxam (Ridomil Gold®)
found with tank mixes of Regalia® with Bacillus subtilis (Sere- were coated with Sepiret® 1171-O on soybean or cotton seeds.
nade®), or Bacillus pumilus (Sonata®), cyprodinil (Vangard®), The treated seeds were seeded in soil infested with R. solani or
or kresoxim-methyl (Sovran®) in non-repeated tests. A syner- P. ultimum. Results show that soybean or cotton seeds coated
gistic effect was also found when Regalia® was applied in with Regalia® had greater or significantly greater emergence
combination with acibenzolar-S-methyl (Actigard®) to control than that of the untreated control. Regalia® showed synergy
lettuce downy mildew. Field trials have confirmed many of when mixed with the synthetic fungicides. Drenching with
Regalia® also significantly increased emergence and growth of
soybean planted in soil infested with R. solani.

Quadris 0.25ug/ml ab
Dip and drench uses
Quadris 0.5ug/ml a We have been testing Regalia® against soil-borne diseases and
Regalia+Quadris 0.25ug/ml c
to increase yield when applied as a pre-transplant dip or in
furrow drench or applied through irrigation. Figure 4 shows
Regalia+Quadris 0.5ug/ml bc the increased feeder root growth that results when Regalia®
is applied, in this case, to strawberry plants in Florida, USA.
Regalia 1:2000 ab
Several treatments were conducted via dip and through irri-
Untreated control a gation; the method of application is not critical, as the root
0 20 40 60 80 100
effects are seen however the product is applied. The grower
Disease severity (%) in this pictured trial produced 300 boxes more per acre than
the chemical or biological competing treatments, resulting in
$20,000 more per acre. He was also able to save water by
Figure 2. Example of Regalia® synergism with a strobilurin fungicide, reducing irrigation due to the large root mass.
azoxystrobin (Quadris®) (Bars with the same letter are not significantly Figure 5 shows data from another crop, processing tomato
different at Fisher’s LSD test at p=0.05 level). Synergy calculation in California, USA, where Regalia® treatments provided an
follows Colby, 1967, E (Efficacy) = % Control > Ee (Expected efficacy) = increased gross return. Additional trials have been conducted
A+B-AB/100, where A and B are the efficacy of the two products. on strawberries in the eastern and western USA, fresh market
potatoes and peppers, and potatoes, all documenting the yield
increases with soil applications.

Regalia 1:2000 b

Regalia 1:2000+Rally c

Regalia 1:1500 c

Regalia 1:1500+Rally c

Rally 40W 0.05ug ai/ml a Other

Regalia biological
Untreated control a product
0 20 40 60 80 100
Disease severity (%)

Figure 3. Example of Regalia® synergism with a triazole fungicide

myclobutanil (Rally®) (Bars with the same letter are not significantly
different at Fisher’s LSD test at p=0.05 level). Synergy calculation
follows Colby, 1967, E (Efficacy) = % Control > Ee (Expected efficacy) = Figure 4. Regalia® root growth after pre-plant dip of strawberry plants
A+B-AB/100, where A and B are the efficacy of the two products. (2010, Plant City, Florida).

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 REGALIA® BIOPROTECTANT
has provided more than disease control but also plant health
effects leading directly to enhanced yield and quality. We have
since explored soil applications, seed treatments, and large
acre applications, all with promising results. Marrone Bio
Innovations has partnered with Syngenta for developing and
marketing Regalia® in Europe, Africa and the Middle East.
The registration submission to the EU occurred in Novem-
ber 2011. FMC is taking the product into Latin America;
Regalia® Maxx was launched in Mexico in November 2011.
We expect rapid global expansion and expansion into more
crops, additional diseases, row crops and soil applications in
the next two years.

References
Bervejillo, J., M.S. Dhatt, & W.D. Gubler. 1999. Evaluation of
fungicides for control of grape powdery mildew. F&N Tests 55:
Figure 5. Increase in gross return on processing tomatoes by
94.
applications of Regalia®.
Bolwell, G.P. & P. Wojtaszek. 1997. Mechanisms for the generation
of reactive oxygen species in plant defence – a broad perspective.
Physiological and Molecular Plant Pathology 51: 347–366.
Bostock, R.M. 2005. Signal crosstalk and induced resistance:
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Colby, S.R. 1967. Calculating synergistic and antagonistic responses
of herbicides combinations. Weeds 15: 20–22.
Daayf, F., A. Schmitt, & R.R. Bélanger. 1997. Evidence of phytoalexin
in cucumber leaves infected with powdery mildew following
treatment with leaf extracts of Reynoutria sachalinensis. Plant
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Daayf, F., M. Ongena, R. Boulanger, I.E. Hadrami, & R.R. Bélanger.
2000. Induction of phenolic compounds in two cultivars of
cucumber by treatment of healthy and powdery mildew-infected
plants with extracts of Reynoutria sachalinensis. Journal of
Chemical and Ecology 26: 1579–1593.
Feys, B.J. & J.E. Parker. 2000. Interplay of signaling pathways in
plant disease resistance. Trends in Genetics 16: 449–455.
Fofana B., D.J. McNally, C.Labbé, R. Boulanger, N. Benhamou,
A. Séguin, & R.R. Bélanger. 2002. Milsana-induced resistance
Figure 6. Example of synergism of Regalia® and Headline® in powdery mildew-infected cucumber plants correlates with
(pyraclostrobin) that results in better yield. the induction of chalcone synthase and chalcone isomerase.
Physiological and Molecular Plant Pathology 61: 121–132.
Fofana B., N. Benhamou, D.J. McNally, C. Labbé, A. Séguin, & R.R.
Uses in large acre crops Bélanger. 2005. Suppression of induced resistance in cucumber
Marrone Bio Innovations has been testing Regalia® for use through disruption of the flavonoid pathway. Phytopathology
in large acre row crops such as wheat, corn and soybean 95: 114–123.
Godard, S., I. Slacanin, O. Viret, & K. Gindro. 2009. Induction
since 2009. We have documented an increase in protein when
of defence mechanisms in grapevine leaves by emodin- and
Regalia® is applied to wheat for Septoria and powdery mildew anthraquinone-rich plant extracts and their conferred resistance to
control, especially in combination with a strobilurin (and downy mildew. Plant Physiology and Biochemistry 47: 827–837.
better than the chemical alone). Figure 6 shows one example Hammerschimdt, R. & J. Kuć. 1982. Liginification as a mechanism
of the yield advantage when Regalia® is applied to soybeans for induced systemic resistance in cucumber. Physiological Plant
as a foliar treatment. Yield is boosted more than the applica- Pathology 20: 61–71.
tion of the chemical treatment alone. This has now been docu- McNally, D., K.V. Wurms, C. Labbé, & R.R. Bélanger. 2003.
mented in multiple growing regions in the United States, and Synthesis of C-glycosyl flavonoid phytoalexins as a site-specific
on corn in 2011 and for three years in soybeans. response to fungal penetration in cucumber. Physiological and
Molecular Plant Pathology 63: 293–303.
Randoux , B., D. Renard, E. Nowak, J. Sanssené, J. Courtois, R.
Durand, & P. Reignault. 2006. Inhibition of Blumeria graminis
Summary and future plans f.sp. tritici germination and partial enhancement of wheat
Since its partial season launch in 2009, and full-year launch defenses by Milsana. Phytopathology 96: 1278–1286.
in 2010, award-winning Regalia® has demonstrated effective Schnee, S., O. Viret, & K. Gindro. 2008. Role of stilbenes in the
control of foliar plant diseases in many fruit, nut and vegeta- resistance of grapevine to powdery mildew. Physiological and
ble crops. The mode of action, Induced Systemic Resistance, Molecular Plant Pathology 72: 128–133.

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 REGALIA® BIOPROTECTANT
Schneider, S. & W.R. Ullrich. 1994. Differential induction of
resistance and enhanced enzyme activities in cucumber and Hai Su is a Senior Scientist at Marrone Bio Innovations, Inc. Davis, Califor-
tobacco caused by treatment with various abiotic and biotic nia. M.S. and Ph.D. degrees were obtained at the Department of Plant Pathol-
inducers. Physiological and Molecular Plant Pathology 45: ogy, China Agricultural Univ. He conducted post-doctoral research on disease
291–304. epidemiology, disease control, and fungicide resistance at Rothamsted Research
Su, H., C. Morgan, B. Campbell, H. Huang, J. Hernandez, M.E. in the UK, Agricultural and Agri-Food Canada, and University of California at
Koivunen, & P.G. Marrone. 2009. Efficacy of new formulations Davis in the U.S.A. He is currently evaluating and developing biofungicides for
of Milsana®, conventional and organic Regalia®, in controlling controlling foliar and soilborne diseases.
cucumber powdery mildew (Sphaerotheca fuliginea). Phyto­
pathology 99:S125 (Abstract). Russell Blair is Marketing Manager at Marrone Bio Innovations and holds a M.S.
Van Loon, L.C., M. Rep, & C.M.J. Pieterse. 2006. Significance of in Plant Biology from Rutgers University, where he also was an Agriculture
inducible defense-related proteins in infected plants. Annual & Resource Management Agent/Assistant Professor. After Rutgers he was a
Review of Phytopathology 44: 135–162. Marketing Manager for Thomas Scientific. At Marrone Bio Innovations, he is
Vance C.P., T.K. Kirk, & R.T. Sherwood. 1980. Lignification responsible for all aspects of Regalia® Product Management.
as a mechanism of disease resistance. Annual Review of
Phytopathology 18: 259–288. Tim Johson is Director of Global Product Development at Marrone Bio Inno-
Věchet, L., J. Martinková, M. Šindelářová, & L. Burketová. 2005. vations. He has a M.S. from Iowa State University and a Ph.D. from Purdue
Compounds of natural origin inducing winter wheat resistance University, both in Entomology. He was with Ecogen Inc for 16 years, hold-
to powdery mildew (Blumeria graminis f.sp. tritici). Plant Soil ing several jobs including Group Leader of Insect Bioassay and Discovery and
Environment 51: 469–475. Director of Commercial Development, where he discovered and commercial-
Wurms, K., C. Labbé, N. Benhamou, & R.R. Bélanger. 1999. ized several Bacillus thuringiensis-based products. After Ecogen he was Manager
Effects of Milsana and benzothiadiazole on the ultrastructure of Commercial Development for the biopesticide company Plato Industries
of powdery mildew haustoria on cucumber. Phytopathology 89: and then joined Marrone Bio Innovations to lead the global field development
728–736. activities.
Zavareh, A.H.J., A.S. Tehrani, & M. Mohammadi. 2007. Effects of
leaf extract of Reynoutria sachalinensis on the defense responses Pamela Marrone is CEO and Founder of Marrone Bio Innovations. She has a
of host in cucumber-powdery mildew interaction. Journal of Ph.D from North Carolina State University in Entomology and started her
Science and Technology of Agriculture and Natural Resources industrial crop protection career at Monsanto Agricultural Company lead-
11: 299–307. ing the Insect Biology Group. After that she was employed by Novo Nordisk
to start a biopesticide subsidiary in Davis, California, Entotech, Inc. She then
founded AgraQuest and was CEO, Chairman and President, discovering and
commercializing biofungicides, Serenade® and Sonata®. She founded Marrone
Bio Innovations in 2006.

Similar articles that appeared in Outlooks on Pest Management include – 2004 15(1) 18; 2004
15(4) 185; 2008 19(1) 24; 2008 19(2) 77; 2010 21(3) 132

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