RegaliaBioprotectantinPlantDiseaseManagement

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An effective biofungicide with novel modes of
action
Article in Pesticide Outlook · October 2002
DOI: 10.1039/b209431m
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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,
knotweed, plant extract, Regalia, Reynoutria sachalinensis, REYSA, SAR
Regalia® as a biofungicide
Plants have long been used as herbal medicines for treatment
of human diseases. However, only a limited number of plant
extracts have been developed into fungicides for plant disease
control. The commercialization of Regalia® illustrates the
potential for plant extracts to be an important tool for disease
management in both organic and conventional production
systems and emphasises the need for additional plant extracts
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
is used as a food in many countries, especially Asia. Knotweed
was formerly formulated as Milsana® in the1980s and was
tested in field trials through the 1990s. Trial demonstrations
were limited to cucumber powdery mildew (Sphaerotheca
fuliginea) and a few other diseases such as Botrytis fruit rot
(Botrytis cinerea) and wheat powdery mildew (Blumeria
graminis f. sp. tritici). Since the 2009 introduction as the
reformulated product Regalia® by Marrone Bio Innovations,
Inc., extensive tests have been conducted in the laboratory,
glasshouse and field on multiple crop-disease systems to evaluate its efficacy for disease control. Test results demonstrate
the efficacy of Regalia® applied as a foliar spray in controlling
a wide range of fungal and bacterial diseases, such as powdery
mildew of cucurbits, downy mildew of lettuce (Bremia lactucae), Botrytis of grapes and strawberries, bacterial spot of
tomatoes and peppers (Xanthomonas campestris pv. vesicatoria), Cercospora on soybeans (Cercospora kikuchii) and
bacterial canker on citrus (Xanthomonas axonopodis pv.
citri), amongst others.
The broad spectrum of disease control by Regalia® relies
on the unique mechanism of induced plant resistance. Studies
in plants show that Regalia® treatment increases the activity
of chalcone synthase and chalcone isomerase in the phenylpropanoid pathway and induces the production and accumulation of phytoalexins. Simple phenolic compounds, which
are fungitoxic, also accumulate. Additional studies show that
Regalia® increases the papillae formation at pathogen penetration 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 fungicides, such as azoles, strobilurins, and sulfur in controlling
powdery mildew and leaf spot diseases, copper in controlling
bacterial diseases, and mancozeb and mefenoxam in controlling 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.
The successful commercialization of Regalia® is the result
of extensive research into efficacy and formulations, demonstration of all application options for efficacy, and innovative
marketing strategies. Integration of plant extracts in disease
management programs can increase yields and quality, reduce
fungicide resistance in pathogens, lower residue of synthetic
fungicides in plants and soil, and protect human health and
the environment.
Mode of action of Regalia®
Reynoutria sachalinensis (Figure 1) (Syn. Polygonum sachalinensis, Fallopia sachalinensis) belongs to the plant family
Polygonaceae and contains anthraquinones such as resveratrol, physicon and emodin, which are known to be antimicrobial. The current formulations available are Regalia® SC
(5% w/w), Regalia® Bioprotectant Concentrate (5% w/w),
and Regalia® Maxx (20% w/w), all of which have better efficacy and broader spectrum than the earlier Milsana® product
and also provide new tools for both conventional and organic
growers (Su et al. 2009). Extensive research results show that
the extract of R. sachalinensis can induce plant resistance by:
Induction of phytoalexins and phenolic compound production;
Increase in production of defense-related proteins;
•
•
•
•
•
•
•
Introduced into North America
as ornamental and for livestock
fodder
Widely distributed invasive,
riparian weed
Insects and birds feed on Reysa
Livestock foraging a control
method
In Buckwheat family, a common
food in Asian cultures
Celery and rhubarb close
relatives
Hybridizes with Japanese
knotweed
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
© 2012 Research Information Ltd. All rights reserved. www.pestoutlook.com
DOI: 10.1564/23feb09
REGALIA® BIOPROTECTANT
Accumulation of reactive oxygen species;
Lignification and papilla formation in cell walls.
Induction of phytoalexin production
Daayf et al. (1997) first demonstrated that elevated phytoalexin and phenolic compounds measured after REYSA
treatment are resposible for the strong resistance of cucumber to powdery mildew (S. fuliginea). Wurms et al. (1999)
also found that increased levels of phenolic compounds
after REYSA treatment increased the resistance of wheat to
powdery mildew (Blumeria graminis f.sp. triciti).
Fofana et al. (2002) discovered increased levels of mRNA
and activity of chalcone synthase (CHS) and chalcone isomerase (CHI), and elevated levels of flavonoid compounds due
to REYSA treatment, which lead to a high level of resistance
in cucumber to powdery mildew. By interrupting the flavonoid pathway in cucumber plants, Fofana et al. (2005) could
down-regulate chalcone synthase in the flavonoid pathway
and reduce resistance to powdery mildew. Similarly, McNally
et al. (2003) further detailed and confirmed that C-glycosyl
flavonoid phytoalexin production after REYSA treatment
increased resistance in cucumber to powdery mildew.
Daayf et al. (2000) reported that the phenolic compounds
ρ-coumaric acid, caffeic acid, ferulic acid and ρ-coumaric
acid methyl ester accumulated after REYSA treatment. These
compounds increased resistance in cucumber to S. fuliginea
in vivo and showed fungitoxic effects on Botrytis cinerea,
Pythium ultimum and P. aphanidermatum.
Additional evidence by Zavareh et al. (2007) showed that
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
flavonoid pathway and it is typically used as an indicator of
resistance to stress, such as disease, drought, and flood.
Pathogenesis-related (PR) proteins
Zavareh et al. (2007) studied the response in cucumber
inoculated and non-inoculated with S. fuliginea after treatment with REYSA. They found that the activity of peroxidase, a plant defense-related protein (van Loon et al., 2006),
increased significantly in similar patterns in treated tissues of
pathogen-inoculated and non-inoculated plants.
Schneider and Ullrich (1994) investigated the mechanism of
REYSA by studying powdery mildew (S. fuliginea) on cucumber and bacterial speck (Pseudomonas syringae pv. tobaci and
P. syringae pv. pisi) on tobacco in parallel. Both cucumber
and tobacco plants treated by REYSA had increased activities
of chitinase, beta-1,3-glucanase (PR-proteins) and PAL, and
resulted in significantly lower disease severity compared with
the untreated control.
Increase of PR proteins and of resistance to powdery
mildew (Blumeria graminis) was also detected in wheat after
treatment with REYSA (Véchet et al., 2005).
Production of reactive oxygen species (ROS)
Among all plant species, a common response to pathogen
infection or physical damage is to generate elevated level of
reactive oxygen species (ROS) such as superoxide radicals
(∙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
in a hypersensitive reaction (HR). ROS also serve as defense
signaling for induced resistance-related pathways (Bolwell &
Wojtaszek, 1997). Thus, ROS are often used as markers for
measuring plant resistance to diseases.
Randoux et al. (2006) found increased accumulation of
reactive oxygen species (ROS) such as H2O2 in wheat after
treatment with REYSA and increased resistance to powdery
mildew. Additionally, Věchet et al. (2005) also treated wheat
with REYSA and after inoculation with powdery mildew
(Blumeria graminis f.sp. triciti) they obtained good disease
control in REYSA treated plants.
Wall apposition
Accumulation of lignin on cell walls helps plants fight against
plant pathogen invasion, and hence is considered another
expression of induced resistance (Vance et al., 1980). For
example, Hammerschimdt and Kuć (1982) found that lignification is responsible for resistance to different pathogens such
as Colletotrichum lagenarium, and Cladosporium cucumerinum in cucumber. Wurms et al. (1999) reported increased
lignification and thickening of cell walls in REYSA-treated
plants. Papillae formed at the penetration sites of the powdery
mildew fungus (S. fuliginea) to prevent penetration of germ
tube of the pathogen. Formation of papillae in REYSA-treated
plants was also confirmed by Fofana et al. (2005).
Additional evidence
Some of the active ingredients such as physcion and emodin
in REYSA and other plant extracts induce resistance in grapevine
It has been shown that REYSA controls powdery mildew
(Uncinula necator) on grapevine (Bervejillo et al., 1999).
Schnee et al. (2008) showed that stilbenic phytoalexin
accumulation is modulating the resistance in grapevine to
powdery mildew. Godard et al. (2009) used Rheum palmatum
(Rhubarb, family Polygonaceae) and Frangula alnus (Alder
Buckthorn, family Rhamnaceae), which contain emodin,
physcion, and other related (anthraquinone-rich) compounds,
to treat grape vines. These plant extracts induced stilbenic
phytoalexin production, increased activity of peroxidase,
induced the hypersensitive reaction (HR), and inhibited spore
germination of Plasmopara viticola (vine downy mildew).
The control of plant pathogens through induced resistance
is universal among various plant and plant pathogen species
(Bostock, 2005; Feys & Parker, 2000). REYSA induces
systemic resistance and works through multiple simultaneous mechanisms in the cellular level, most important of which
lead to increase in phytoalexins, phenolics, PR-proteins, reactive oxygen species, and cell wall lignification.
Synergy
Combination (tank mix) of Regalia® and other commercial
fungicides can be an effective and efficient measure in increasing
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REGALIA® BIOPROTECTANT
pathogen control efficacy and managing fungicide resistance
in pathogens. Greenhouse and growth chamber experiments
were conducted to evaluate the efficacy of Regalia® in combination with commonly used chemical and biological fungicides in controlling powdery mildew on cucurbits and lettuce
downy mildew. The results show a statistically significant
synergistic effect with Regalia® in a tank mix with azoxystrobin (Quadris®), myclobutanil (Rally® 40W), quinoxyfen
(Quintec®), or triflumizole (Procure®), and other commercial
fungicides in controlling powdery mildew on cucumber in
repeated tests. Only an additive effect with no synergy was
found with tank mixes of Regalia® with Bacillus subtilis (Serenade®), or Bacillus pumilus (Sonata®), cyprodinil (Vangard®),
or kresoxim-methyl (Sovran®) in non-repeated tests. A synergistic effect was also found when Regalia® was applied in
combination with acibenzolar-S-methyl (Actigard®) to control
lettuce downy mildew. Field trials have confirmed many of
Quadris 0.25ug/ml
ab
a
c
bc
Regalia+Quadris 0.5ug/ml
Regalia 1:2000
ab
a
Untreated control
0
20
40
60
80
100
Disease severity (%)
Figure 2. Example of Regalia® synergism with a strobilurin fungicide,
azoxystrobin (Quadris®) (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) =
A+B-AB/100, where A and B are the efficacy of the two products.
Regalia 1:2000
Seed treatment uses
Regalia® can also be applied as a seed coating or drenched
in soil to control soilborne diseases caused by Rhizoctonia
solani or Pythium ultimum in soybean and cotton. In our
studies, Regalia® alone, or mixed with azoxystrobin (Quadris®), fludioxonil (Scholar®), or mefenoxam (Ridomil Gold®)
were coated with Sepiret® 1171-O on soybean or cotton seeds.
The treated seeds were seeded in soil infested with R. solani or
P. ultimum. Results show that soybean or cotton seeds coated
with Regalia® had greater or significantly greater emergence
than that of the untreated control. Regalia® showed synergy
when mixed with the synthetic fungicides. Drenching with
Regalia® also significantly increased emergence and growth of
soybean planted in soil infested with R. solani.
Dip and drench uses
Quadris 0.5ug/ml
Regalia+Quadris 0.25ug/ml
these results found in the greenhouse tests. The examples of
synergy with azoxystrobin and myclobutanil in a greenhouse
trial are shown in Figure 2 and 3.
We have been testing Regalia® against soil-borne diseases and
to increase yield when applied as a pre-transplant dip or in
furrow drench or applied through irrigation. Figure 4 shows
the increased feeder root growth that results when Regalia®
is applied, in this case, to strawberry plants in Florida, USA.
Several treatments were conducted via dip and through irrigation; the method of application is not critical, as the root
effects are seen however the product is applied. The grower
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
reducing irrigation due to the large root mass.
Figure 5 shows data from another crop, processing tomato
in California, USA, where Regalia® treatments provided an
increased gross return. Additional trials have been conducted
on strawberries in the eastern and western USA, fresh market
potatoes and peppers, and potatoes, all documenting the yield
increases with soil applications.
b
Regalia 1:2000+Rally
c
Regalia 1:1500
c
Regalia 1:1500+Rally
c
Rally 40W 0.05ug ai/ml
a
Untreated control
Regalia
a
0
20
40
60
80
Other
biological
product
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) =
A+B-AB/100, where A and B are the efficacy of the two products.
Figure 4. Regalia® root growth after pre-plant dip of strawberry plants
(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 November 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
Figure 5. Increase in gross return on processing tomatoes by
applications of Regalia®.
Figure 6. Example of synergism of Regalia® and Headline®
(pyraclostrobin) that results in better yield.
Uses in large acre crops
Marrone Bio Innovations has been testing Regalia® for use
in large acre row crops such as wheat, corn and soybean
since 2009. We have documented an increase in protein when
Regalia® is applied to wheat for Septoria and powdery mildew
control, especially in combination with a strobilurin (and
better than the chemical alone). Figure 6 shows one example
of the yield advantage when Regalia® is applied to soybeans
as a foliar treatment. Yield is boosted more than the application of the chemical treatment alone. This has now been documented in multiple growing regions in the United States, and
on corn in 2011 and for three years in soybeans.
Summary and future plans
Since its partial season launch in 2009, and full-year launch
in 2010, award-winning Regalia® has demonstrated effective
control of foliar plant diseases in many fruit, nut and vegetable crops. The mode of action, Induced Systemic Resistance,
Bervejillo, J., M.S. Dhatt, & W.D. Gubler. 1999. Evaluation of
fungicides for control of grape powdery mildew. F&N Tests 55:
94.
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:
Straddling the line between cost and benefit. Annual Review of
Phytopathology 43: 545–80.
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
Physiology 113: 719–727.
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
in powdery mildew-infected cucumber plants correlates with
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.
Bélanger. 2005. Suppression of induced resistance in cucumber
through disruption of the flavonoid pathway. Phytopathology
95: 114–123.
Godard, S., I. Slacanin, O. Viret, & K. Gindro. 2009. Induction
of defence mechanisms in grapevine leaves by emodin- and
anthraquinone-rich plant extracts and their conferred resistance to
downy mildew. Plant Physiology and Biochemistry 47: 827–837.
Hammerschimdt, R. & J. Kuć. 1982. Liginification as a mechanism
for induced systemic resistance in cucumber. Physiological Plant
Pathology 20: 61–71.
McNally, D., K.V. Wurms, C. Labbé, & R.R. Bélanger. 2003.
Synthesis of C-glycosyl flavonoid phytoalexins as a site-specific
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
f.sp. tritici germination and partial enhancement of wheat
defenses by Milsana. Phytopathology 96: 1278–1286.
Schnee, S., O. Viret, & K. Gindro. 2008. Role of stilbenes in the
resistance of grapevine to powdery mildew. Physiological and
Molecular Plant Pathology 72: 128–133.
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REGALIA® BIOPROTECTANT
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resistance and enhanced enzyme activities in cucumber and
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Hai Su is a Senior Scientist at Marrone Bio Innovations, Inc. Davis, California. M.S. and Ph.D. degrees were obtained at the Department of Plant Pathology, China Agricultural Univ. He conducted post-doctoral research on disease
epidemiology, disease control, and fungicide resistance at Rothamsted Research
in the UK, Agricultural and Agri-Food Canada, and University of California at
Davis in the U.S.A. He is currently evaluating and developing biofungicides for
controlling foliar and soilborne diseases.
Russell Blair is Marketing Manager at Marrone Bio Innovations and holds a M.S.
in Plant Biology from Rutgers University, where he also was an Agriculture
& Resource Management Agent/Assistant Professor. After Rutgers he was a
Marketing Manager for Thomas Scientific. At Marrone Bio Innovations, he is
responsible for all aspects of Regalia® Product Management.
Tim Johson is Director of Global Product Development at Marrone Bio Innovations. He has a M.S. from Iowa State University and a Ph.D. from Purdue
University, both in Entomology. He was with Ecogen Inc for 16 years, holding several jobs including Group Leader of Insect Bioassay and Discovery and
Director of Commercial Development, where he discovered and commercialized several Bacillus thuringiensis-based products. After Ecogen he was Manager
of Commercial Development for the biopesticide company Plato Industries
and then joined Marrone Bio Innovations to lead the global field development
activities.
Pamela Marrone is CEO and Founder of Marrone Bio Innovations. She has a
Ph.D from North Carolina State University in Entomology and started her
industrial crop protection career at Monsanto Agricultural Company leading 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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