doi: 10.3389/fmicb.2016.00548
Edited by:
Daniela Gwiazdowska, Poznan University of Economics, Poland
Reviewed by:
Jose M. Diaz-Minguez, CIALE - Universidad de Salamanca, Spain Jon Y. Takemoto, Utah State University, USA
*Correspondence:
Wentzel C. A. Gelderblom gelderblomw@cput.ac.za
Specialty section:
This article was submitted to Fungi and Their Interactions, a section of the journal Frontiers in Microbiology
Received: 28 January 2016 Accepted: 04 April 2016 Published: 26 April 2016 Citation:
Alberts JF, van Zyl WH and Gelderblom WCA (2016) Biologically Based Methods for Control of Fumonisin-Producing Fusarium Species and Reduction of the Fumonisins. Front. Microbiol. 7:548. doi: 10.3389/fmicb.2016.00548
Biologically Based Methods for
Control of Fumonisin-Producing
Fusarium
Species and Reduction of
the Fumonisins
Johanna F. Alberts
1, Willem H. van Zyl
2and Wentzel C. A. Gelderblom
1*
1Mycotoxicology and Chemoprevention Research Group, Institute of Biomedical and Microbial Biotechnology, Cape Peninsula University of Technology, Bellville, South Africa,2Microbiology Department, Stellenbosch University, Stellenbosch, South Africa
Infection by the fumonisin-producing Fusarium spp. and subsequent fumonisin
contamination of maize adversely affect international trade and economy with deleterious
effects on human and animal health. In developed countries high standards of the major
food suppliers and retailers are upheld and regulatory controls deter the importation
and local marketing of fumonisin-contaminated food products. In developing countries
regulatory measures are either lacking or poorly enforced, due to food insecurity,
resulting in an increased mycotoxin exposure. The lack and poor accessibility of
effective and environmentally safe control methods have led to an increased interest
in practical and biological alternatives to reduce fumonisin intake. These include the
application of natural resources, including plants, microbial cultures, genetic material
thereof, or clay minerals pre- and post-harvest. Pre-harvest approaches include breeding
for resistant maize cultivars, introduction of biocontrol microorganisms, application
of phenolic plant extracts, and expression of antifungal proteins and fumonisin
degrading enzymes in transgenic maize cultivars. Post-harvest approaches include
the removal of fumonisins by natural clay adsorbents and enzymatic degradation of
fumonisins through decarboxylation and deamination by recombinant carboxylesterase
and aminotransferase enzymes. Although, the knowledge base on biological control
methods has expanded, only a limited number of authorized decontamination products
and methods are commercially available. As many studies detailed the use of natural
compounds in vitro, concepts in reducing fumonisin contamination should be developed
further for application in planta and in the field pre-harvest, post-harvest, and during
storage and food-processing. In developed countries an integrated approach, involving
good agricultural management practices, hazard analysis and critical control point
(HACCP) production, and storage management, together with selected biologically
based treatments, mild chemical and physical treatments could reduce fumonisin
contamination effectively. In rural subsistence farming communities, simple, practical,
and culturally acceptable hand-sorting, maize kernel washing, and dehulling intervention
methods proved to be effective as a last line of defense for reducing fumonisin exposure.
Biologically based methods for control of fumonisin-producing Fusarium spp. and
decontamination of the fumonisins could have potential commercial application, while
simple and practical intervention strategies could also impact positively on food safety
and security, especially in rural populations reliant on maize as a dietary staple.
Keywords: Fusarium, fumonisins, prevention, biological control, reduction, sub-Saharan countries
INTRODUCTION
Fusarium
spp. are agriculturally important plant pathogenic
fungi
associated with disease and mycotoxin contamination of
grain crops (
Wild and Hall, 2000; Picot et al., 2011
). Fusarium
ear rot in maize is one of the major diseases affecting maize
production worldwide and poses an enormous threat to the
international trade of foods and feeds. Fungal species of Fusarium
Section Liseola, including Fusarium verticillioides, Fusarium
proliferatum, and Fusarium subglutinans are some of the most
important causative fungal agents of Fusarium ear or kernel
rot as well as symptomless infection of maize crops, leading to
contamination with the fumonisin mycotoxins (
Munkvold et al.,
1997
).
Fifteen Fusarium spp. have been reported to produce
fumonisins. Eight species are from the Section Liseola, i.e.,
F. verticilloides, Fusarium sacchari, Fusarium fujikuroi, F.
proliferatum, F. subglutinans, Fusarium thapsinum, Fusarium
anthophilum, and Fusarium globosum (
Rheeder et al., 2002
).
Another five species fall within Section Dlaminia, i.e., Fusarium
nygamai, Fusarium dlamini, and Fusarium napiforme. Trace
amounts of fumonisin were detected in culture material of two
species, i.e., Fusarium andiyazi and Fusarium pseudonygamai.
The remaining two fumonisin-producing Fusarium spp. are
one species in Section Elegans, i.e., Fusarium oxysporum and
one in Section Arthrosporiella, i.e., Fusarium polyphialidicum.
The fumonisins are associated with several diseases in humans,
animals, poultry, and fish (
Marasas, 2001; Marasas et al., 2004;
Kimanya et al., 2010
) and are classified as Group 2B carcinogens
(
IARC, 2002
). Home-grown maize is a major dietary staple in
southern Africa and known to be frequently contaminated with
unacceptable levels of fumonisins, with fumonisin B
1(FB
1) being
the most prevalent natural occurring fumonisin (
Marasas, 2001;
Marasas et al., 2004; Shephard et al., 2007, 2013; Burger et al.,
2010
). The Eastern Cape Province of South Africa is one of the
areas in the world where the highest levels of FB
1were recorded
in home-grown maize. As a result exposure to FB
1in adults is
more than four times above the provisional maximum tolerable
daily intake (2 µg FB
1/kg body weight/day) set by the Joint Food
and Agriculture Organization of the United Nations and the
World Health Organization (FAO/WHO) Expert Committee on
Food Additives (
Bolger et al., 2001
).
The fumonisins comprise a group of 28 characterized analogs,
which can be separated into four main groups: fumonisin A,
B, C, and P (
Rheeder et al., 2002
). The fumonisin B (FB)
analogs, which includes FB
1, FB
2, and FB
3, are the most abundant
naturally occurring fumonisins, with FB
1predominating and
usually being found at the highest levels. Apart from FB, some
of the other analogs may occur in naturally contaminated maize
at relatively low levels. The complete fumonisin molecule plays
an important role in toxic and cancer-initiating activities in vivo
(
Gelderblom et al., 1993
). Studies evaluating the
structure-activity relationship of fumonisin analogs, hydrolysis products
and a monomethyl ester of FB
1in short-term carcinogenesis in
rats and cytotoxicity assays in primary rat hepatocytes, indicated
that the free amino group plays a pivotal role in the toxicological
effects of the fumonsins in vitro and in vivo. It was suggested
that the tricarballylic acid moiety is required for effective
absorption of the fumonisins from the gut. The fumonisins
disrupt sphingolipid biosynthesis by inhibiting the enzyme
ceramide synthase (
Wang et al., 1991
), and the tricarballylic acid
moiety is required for maximal effect (
Van der Westhuizen et al.,
1998
).
Fusarium infect maize in the field with the highest levels of
fumonisins present at harvest, concentrated in the pericarp and
embryo of the maize kernel (
Fandohan et al., 2006; Kimanya
et al., 2008; Burger et al., 2013
). Kinetics of Fusarium growth
and mycotoxin production are mainly affected by water activity,
temperature, and atmospheric composition, while nutritional
factors such as kernel endosperm composition and nitrogen
sources also play an important role (
Chulze, 2010; Picot et al.,
2011
). Fumonisin production strongly depends on the kernel
stage, and may be regulated by physicochemical factors that vary
during ear ripening. Insect damage of maize by the European
corn borer (Ostrinia nubilalis Hübner) and the corn earworm
(Helicoverpa zea Boddie) further favors Fusarium infection (
Betz
et al., 2000
).
Methods for reduction
of fumonisins in maize are applied
pre-harvest or during harvesting and processing (
Wild and
Gong, 2010
). These include several existing strategies to
reduce Fusarium growth and production of fumonisins in
food sources, i.e., controlled agricultural practices, ensiling
strategies, breeding for insect and fungal resistance in maize
cultivars, various physical-, chemical-, and biological treatment
methods and genetic engineering approaches. Good agricultural
management and hazard analysis and critical control point
(HACCP) practices promote the general condition of crops,
reducing but not eliminating fungal growth, and mycotoxin
contamination, while resistance breeding strives to achieve a
balance between developing resistant crops and maintaining
high quality crop yield (
Cleveland et al., 2003; Wild and
Gong, 2010
). However, optimization of agricultural management
practices is not always possible due to high production costs, the
geographical location or nature of the production systems, and
challenging environmental conditions.
Several physical and chemical control methods for mycotoxins
have been commercialized involving sorting and flotation,
solvent extraction, chemical detoxification by alkalization (e.g.,
ammonia, sodium hydroxide, and sulfur dioxide treatments),
oxidation (e.g., ozone), and irradiation and pyrolysis (
He and
Zhou, 2010
). There are, however, several limitations, challenges,
and concerns with regards to physical and chemical control
methods (
Schatzmayr et al., 2006
). Physical methods generally
have low efficacy and less specificity, while chemical methods are
not always effective, are considered expensive and may decrease
the nutritional value of foods, affect the sensory quality, and
could produce toxic derivatives (
Alabouvette et al., 2009; He
and Zhou, 2010
). Furthermore, methods involving fungicides
pose a potential health, safety, and environmental risk as certain
antifungal chemical compounds are not biodegradable or have
a long degradation period, could contaminate soil and water
and their effect on food quality and human health is a concern
(
Larkin and Fravel, 1998; da Cruz Cabral et al., 2013
). Prolonged
chemical treatment of grains can lead to the development of
resistance in fungal strains, a demand for higher concentrations,
and an increase in toxic residues in food crops. Increasingly
more stringent regulation is enforced with regards to the use
of chemical control methods together with a strong consumer
demand to reduce the use of potentially harmful chemicals in
the food supply (
Liu et al., 2013
). There is also an ecological
and societal movement toward safe and natural food, without
chemical treatments and/or preservatives (
Edlayne et al., 2009
).
Research over the past 25 years indicates support for
agricultural management practices and a renewed interest
in practical and biological control methods as possible
alternatives. In this regard several methods for controlling
fungal growth and mycotoxin production pre- and
post-harvest involving clay minerals, plant extracts and a variety
of microbial taxa have been commercialized (
He and Zhou,
2010
). In rural subsistence farming communities a number
of effective, practical, and culturally acceptable intervention
methods have been developed (
Kimanya et al., 2008; Van
der Westhuizen et al., 2010
). While the focus in the past
was more on the most economically important mycotoxins,
i.e., aflatoxin B
1(AFB
1), much less information is available
on other important mycotoxins such as FB
1, trichothecenes,
zearalenone, citrinin, and patulin (
Kabak et al., 2006
). This
paper presents a comprehensive overview of recent research
on biological- and practical-based approaches for control of
fumonisin-producing Fusarium spp. and methods for reduction
thereof during pre- and post-harvest conditions. Current
information on the application of natural clay adsorbents,
biocontrol organisms, antioxidants, essential oils, plant extracts,
and molecular approaches are reviewed; as well as practical
and culturally acceptable methods for reduction of fumonisin
exposure in rural subsistence farming communities.
PRE-HARVEST BIOLOGICALLY BASED
CONTROL METHODS FOR
FUMONISIN-PRODUCING FUSARIUM Spp.
Biocontrol Microorganisms
This approach involves a three-way interaction between the
host commodity, the pathogen and the antagonistic biocontrol
microorganism together with dynamics such as competition
for nutrients and space, parasitism of the pathogen, secretion
of antifungal compounds, induction of systemic resistance
(ISR), biofilm formation and involvement with reactive
oxygen species in defense response (
Larkin and Fravel, 1998;
Alabouvette et al., 2009
). Recent research also suggested that the
aflatoxin biocontrol mechanism, employing atoxigenic strains of
Aspergillus flavus, is triggered by physical contact or interaction
between hyphae of the competing fungal strains (
Damann, 2014
).
Essential criteria for effective biocontrol microorganisms include
the ability to colonize the plant part infected by the pathogen
organism, efficacy under the relevant environmental conditions
and compatibility with other control methods that are applied
(
Bacon and Hinton, 2011; Liu et al., 2013
). Niche overlap indices
(NOIs) provide information on ecological similarity, coexistence,
and competition between microorganisms in a specific niche
and assists in identifying possible microbial antagonists
against F. verticillioides colonization (
Cavaglieri et al., 2004
).
Microorganisms naturally associated with and adapted to the
vegetative parts of a specific plant, sharing the ecological niche
with pathogen microorganisms, could hold advantages as
biocontrol agents. One such a microorganism, Bacillus subtilis
occupies the same ecological niche as F. verticillioides within
the maize plant and effectively inhibits growth of the fungus,
based on competitive exclusion (
Bacon et al., 2001
; Table 1).
B. subtilis is considered generally regarded as safe (GRAS) by
the United States Food and Drug Administration [US FDA,
GRAS substances evaluated by the Select Committee on GRAS
substances (SCOGS)], is easy to cultivate and manipulate
genetically, and therefore suitable for industrial processes. A
pre-harvest biological control system, involving B. subtilis RRC101,
was developed on maize which reduces fumonisin accumulation
during the endophytic growth phase of F. verticillioides (= F.
moniliforme;
Bacon et al., 2001
). The endophytic phase of F.
verticillioides is transferred vertically to the next generation
through clonal infection of seeds. This phase is characterized
by intercellular systemic infection of plants and seeds, which
cannot be controlled with fungicides. Effective biocontrol has
also been demonstrated with wild type and fusaric acid resistant
mutant strains of the bacterial endophyte, Bacillus mojavensis,
in vitro and in planta (
Bacon and Hinton, 2011
). Efficacy of these
strains under field conditions could be influenced by fusaric acid
produced by F. verticillioides. The mechanism of biocontrol by
B. mojavensis is complex and still unclear, as indicated by broad
differences in maize seedling protection by a range of strains
evaluated.
Pediococcus pentosaceus, a lactic acid bacterial isolate from
maize, inhibits F. verticillioides and F. proliferatum growth
in vitro (
Dalie et al., 2010
; Table 1). Antifungal activity in P.
pentosaceus culture supernatant was observed toward the end
of the exponential phase of growth and was pH dependent.
The antifungal metabolites produced proved to be heat stable
and resistant to proteolytic enzymes. Culture fractions exhibiting
antifungal activity contained compounds with molecular masses
ranging from 500 to 1400 Da. P. pentosaceus has GRAS status,
has been widely used in the fermentation of a variety of foods
and could be suitable as biocontrol organism to improve the
quality of ensilage. Clonostachys rosae, a fungal isolate from
straw, stubble, seed surfaces, and the phylosphere or roots of
cereal crops, effectively reduced sporulation of F. verticillioides
and F. proliferatum on maize stalks in vitro and in field trials
T A B L E 1 | C u rr e n t in fo rma ti o n o n re d u c ti o n o f fu mo n is in -p ro d u c in g F u s a ri u m s p p . b y b io c o n tr o l mi c ro o rg a n is ms in v it ro , in p la n ta , a n d in fi e ld tr ia ls . B io c o n tr o l mi c ro o rg a n is m F u s a ri u m s p p . s tu d ie d Te s t s y s te m w it h d e ta il s o f e x p e ri me n ta l mo d e l R e d u c ti o n c ri te ri a A p p li c a ti o n R e fe re n c e (s ) Tr ic h o d e rm a sp p . st ra in s a g g re ss iv e to w a rd F u s a ri u m ve rt ic ill io id e s (= m o n ili fo rm e ): Tr ic h o d e rm a h a rz ia n u m T 1 a n d T 2 , Tr ic h o d e rm a vi ri d e T 5 a n d T 6 F. ve rt ic ill io id e s (= F. m o n ili fo rm e ) In vi tr o st u d ie s o n th e p o te n tia lf o r b io lo g ic a lc o n tr o lo f A s p e rg ill u s fla vu s a n d F. m o n ili fo rm e b y Tr ic h o d e rm a sp p .: a st u d y o f th e p ro d u c tio n o f e xt ra c e llu la r m e ta b o lit e s b y Tr ic h o d e rm a sp p . In vi tr o : E ff e c t o f c a rb o n so u rc e o n a n tif u n g a l p ro p e rt ie s o f Tr ic h o d e rm a sp p .; A n tif u n g a la c tiv iti e s o f Tr ic h o d e rm a sp p . c u ltu re fil tr a te s: p re p a ra tio n o f liq u id c u ltu re fil tr a te s; P D A p la te a ss a y; d e te rm in a tio n o f in h ib iti o n ; sc a n n in g e le c tr o n m ic ro sc o p y o f m yc e lia lp lu g s; P ro d u c tio n o f vo la til e s: in ve rt e d fu n g a l c u ltu re s; c o lo n y d ia m e te rs ; P ro d u c tio n o f e xt ra c e llu la r e n zy m e s: a g a r p la te m e th o d ; m e a su re m e n t o f d e p le tio n o f n u tr ie n t so u rc e s; E va lu a tio n o f o sm o tic p o te n tia l; P ro d u c tio n o f a n tib io tic s: so lid a g a r p la te a ss a y; m o n ito rin g zo n e s o f in h ib iti o n o f E . c o li a n d S ta p h yl o c o c c u s a u re u s In vi tr o : E ff e c t o f c a rb o n so u rc e o n a n tif u n g a lp ro p e rt ie s o f Tr ic h o d e rm a sp p .: Tr ic h o d e rm a sp p . in h ib ite d F. ve rt ic ill io id e s g ro w th o n g ro w th m e d iu m w ith g lu c o se a s c a rb o n so u rc e ; n o in h ib iti o n w ith su c ro se a s c a rb o n so u rc e ; in h ib iti o n o b se rv e d w ith L -a la n in e a s n itr o g e n so u rc e ; T. h a rz ia n u m T 2 a n d T. vi ri d e T 5 e xh ib ite d th e st ro n g e st in h ib ito ry e ff e c t; A n tif u n g a la c tiv iti e s o f Tr ic h o d e rm a sp . c u ltu re fil tr a te s: g e n e ra li n h ib iti o n o f F. ve rt ic ill io id e s g ro w th ; c u ltu re fil tr a te s o f T. h a rz ia n u m T 2 a n d T. vi ri d e T 5 re su lte d in p ro n o u n c e d m o rp h o lo g ic a la lte ra tio n s; P ro d u c tio n o f vo la til e s: th e p re se n c e o f vo la til e c o m p o u n d s o f T. h a rz ia n u m T 2 , T. vi ri d e T 5 a n d T 6 w e re a b le to su p p re ss F. ve rt ic ill io id e s g ro w th ; P ro d u c tio n o f e xt ra c e llu la r e n zy m e s: a m yl a se a n d c e llu lo se a c tiv ity e xh ib ite d b y a ll fo u r st ra in s; ly p o ly tic a c tiv ity e xh ib ite d b y T. h a rz ia n u m T 1 , T 2 a n d T. vi ri d e T 5 ; p ro te o ly tic a c tiv ity e xh ib ite d b y T. h a rz ia n u m T 2 a n d T. vi ri d e T 5 ; e xt ra c e llu la r p e c tin o ly tic a c tiv ity e xh ib ite d b y T. h a rz ia n u m T 1 a n d T. vi ri d e T 5 . T. vi ri d e p ro d u c e d th e w id e st sp e c tr u m o f e xt ra c e llu la r e n zy m e s; E va lu a tio n o f o sm o tic p o te n tia l: e n zy m e p ro d u c tio n d e c re a se d w ith in c re a si n g o sm o tic p o te n tia l; P ro d u c tio n o f a n tib io tic s: T. vi ri d e T 5 e xh ib ite d th e g re a te st in h ib ito ry e ff e c t o n E . c o li a n d S . a u re u s , su g g e st in g p ro d u c tio n o f a n tib io tic s Tr ic h o d e rm a sp p . e xh ib ite d p o te n tia lf o r b io c o n tr o la g a in st m yc o to xi n -p ro d u c in g fu n g i; th e la c k o f o sm o to le ra n c e in a ir-d rie d se e d c o u ld b e a d is a d va n ta g e C a lis tr u e t a l., 1 9 9 7 T. vi ri d e U P S 1 0 1 is o la te d fr o m ro o t se g m e n ts o f c o rn p la n ts g ro w n in P ie d m o n t, G e o rg ia , U S A F. ve rt ic ill io id e s (= F. m o n ili fo rm e ) st ra in s: R R C P A T, R R C P A T g u s T. vi ri d e su p p re ss e s F B1 p ro d u c tio n b y F. m o n ili fo rm e . In vi tr o : A n tif u n g a la c tiv ity : si n g le a n d c o -c u lti va tio n o n P D A ; c o lo n y d ia m e te rs ; E ff e c t o n F B1 le ve ls : si n g le a n d c o -c u lti va tio n o n m a iz e ke rn e ls ; d e te rm in a tio n o f F B1 le ve ls In vi tr o : A n tif u n g a la c tiv ity : T. vi ri d e su p p re ss e d ra d ia l e xt e n si o n o f F. ve rt ic ill io id e s c o lo n ie s (4 6 % re d u c tio n a ft e r 6 d a ys ; 9 0 % a ft e r 1 4 d a ys ); E ff e c t o n F B1 le ve ls : T. vi ri d e su p p re ss e d F B1 p ro d u c tio n b y F. ve rt ic ill io id e s w h e n c o -c u lti va te d o n m a iz e ke rn e ls ; 8 5 % re d u c tio n in F B1 le ve ls w h e n th e T. vi ri d e a n d F. ve rt ic ill io id e s w e re in o c u la te d si m u lta n e o u sl y; 7 2 % re d u c tio n in F B1 le ve ls w h e n T. vi ri d e w a s in o c u la te d 7 d a ys a ft e r F. ve rt ic ill io id e s Tr ic h o d e rm a sp p . m a in ly a p p lie d to so il a s b io c o n tr o la g e n ts ; T. vi ri d e c o u ld b e a p p lie d to in h ib it F. ve rt ic ill io id e s g ro w th p re -h a rv e st , to p re ve n t d is e a se d u rin g p la n t d e ve lo p m e n t, p o st h a rv e st d u rin g st o ra g e o r to su p p re ss F B1 a c c u m u la tio n in in a d e q u a te ly d rie d m a iz e ke rn e ls ; a p p lic a b le fo r F B1 re d u c tio n in m a iz e ke rn e ls in te n d e d fo r a n im a lf e e d Y a te s e t a l., 1 9 9 9 (C o n ti n u e d )
T A B L E 1 | C o n ti n u e d B io c o n tr o l mi c ro o rg a n is m F u s a ri u m s p p . s tu d ie d Te s t s y s te m w it h d e ta il s o f e x p e ri me n ta l mo d e l R e d u c ti o n c ri te ri a A p p li c a ti o n R e fe re n c e (s ) B a c ill u s s u b ti lis st ra in s is o la te d fr o m m a iz e in n o rt h e rn It a ly : B . s u b ti lis R R C 1 0 1 (w ild ty p e ) (P a te n t 5 , 9 9 4 , 1 1 7 ), B . s u b ti lis R R C 2 6 ss a n d B . s u b ti lis R R C 2 4 w f (r ifa m p ic in re si st a n t m u ta n ts ); F. ve rt ic ill io id e s (= m o n ili fo rm e ) W ild ty p e st ra in s: M R C 8 2 6 , R R C 4 1 0 , R R C P A T, R R C 4 0 8 ; F. ve rt ic ill io id e s tr a n sf o rm e d e c o lo g ic a lm a rk e r st ra in s: M R C 8 2 6 g u s, R R C P A T g u s, R R C 4 0 8 g u s B io lo g ic a lc o n tr o lo f F. m o n ili fo rm e in m a iz e . In p la n ta : Y o u n g a n d vi g o ro u s m a iz e se e d lin g s: P la n t p o t c u ltu re s su b je c te d to d ro u g h t tr e a tm e n ts ; se e d tr e a te d w ith B . s u b ti lis ; p la n ts c u lti va te d in so il in fe st e d w ith F. ve rt ic ill io id e s ; p la n t g ro w th lig h t ro o m ; d e te rm in a tio n o f se e d lin g h e ig h t a n d b la d e w id th ; p e rc e n ta g e se e d lin g ro o t in fe c tio n ; C F U c o u n ts o f B . s u b ti lis a n d F. ve rt ic ill io id e s in so il; d e te rm in a tio n o f F B1 le ve ls ; M a tu re m a iz e p la n ts : Te n w e e k o ld m a iz e p la n ts ; d e te rm in a tio n o f F B1 le ve ls in ro o ts , st e m s, le a ve s a n d ke rn e ls In p la n ta : Y o u n g a n d vi g o ro u s m a iz e se e d lin g s: B . s u b ti lis e xh ib ite d a p ro te c tiv e e ff e c t o n m a iz e se e d lin g g ro w th a n d p e rc e n ta g e se e d lin g ro o t in fe c tio n ; B . s u b ti lis re d u c e d F. ve rt ic ill io id e s c o lo n iz a tio n o f so ils ; F B1 w a s si g n ific a n tly re d u c e d (5 0 % ) b y a ll b a c te ria l tr e a tm e n ts , e sp e c ia lly u n d e r d ro u g h t st re ss ; M a tu re m a iz e p la n ts : B . s u b ti lis e xh ib ite d p ro te c tio n in m a tu re d p la n ts a t th e ke rn e lfil ls ta g e B . s u b ti lis c o u ld b e a p p lie d a s se e d tr e a tm e n t to a c t a s b io c o n tr o la g e n t d u rin g th e g ro w th o f m a iz e p la n ts ; e va lu a tio n o f B . s u b ti lis u n d e r fie ld c o n d iti o n s n e e d e d B a c o n e t a l., 2 0 0 1 A la rg e va rie ty o f p o te n tia la n ta g o n is tic b a c te ria la n d fu n g a l st ra in s is o la te d fr o m st ra w , st u b b le , se e d su rf a c e s, a n d th e p h yl o sp h e re o r ro o ts o f c e re a lc ro p s; A d d iti o n a li so la te s: C h a e to m iu m sp p ., F u s a ri u m e q u is e ti F u s a ri u m is o la te s fr o m in fe c te d w h e a t g ra in s in T h e N e th e rla n d s: F u s a ri u m c u lm o ru m , F u s a ri u m g ra m in e a ru m , F u s a ri u m p ro lif e ra tu m , F. ve rt ic ill io id e s P o te n tia lo f fu n g a la n ta g o n is ts fo r b io -c o n tr o lo f F u s a ri u m sp p . in w h e a t a n d m a iz e th ro u g h c o m p e tit io n in c ro p d e b ris . In vi tr o : R e d u c tio n o f F u s a ri u m sp p . c o n id ia fo rm a tio n : w h e a t st ra w b io a ss a y; p re -i n o c u la tio n o f st ra w w ith F u s a ri u m sp p .; su b se q u e n t in o c u la tio n o f st ra w w ith p o te n tia la n ta g o n is ts ; d e te rm in a tio n o f th e n u m b e r o f c o n id ia : m ic ro sc o p y b io a ss a y; R e d u c tio n o f F u s a ri u m sp p . c o n id ia fo rm a tio n : m a iz e st u b b le b io a ss a y; p ro c e d u re s si m ila r to th e w h e a t st ra w b io a ss a y; F ie ld tr ia ls : M a iz e st a lk s: d e te rm in a tio n o f a n ta g o n is m ; p re -i n o c u la tio n o f st a lk s w ith p o te n tia l a n ta g o n is ts ; su b se q u e n t in o c u la tio n o f st a lk s w ith F. ve rt ic ill io id e s , F. p ro lif e ra tu m a n d F. g ra m in e a ru m ; p lo ts in o c u la te d w ith st rip s c o n ta in in g st a lk p ie c e s; c u ltu rin g o f h a rv e st e d st a lk s o n m o d ifie d P D A ; id e n tific a tio n o f F. ve rt ic ill io id e s , F. p ro lif e ra tu m a n d F. g ra m in e a ru m : c o lo n y m o rp h o lo g y a n d m ic ro sc o p ic e xa m in a tio n ; In vi tr o : R e d u c tio n o f F u s a ri u m sp p . c o n id ia fo rm a tio n (w h e a t st ra w b io a ss a y) : sp o ru la tio n o f F. c u lm o ru m a n d F. g ra m in e a ru m o n st ra w : o ve ra ll re d u c tio n (> 8 0 % ) b y a n ta g o n is tic is o la te s; S p o ru la tio n o f F. c u lm o ru m o n C lo n o s ta c h ys ro s e a -t re a te d st ra w : 8 5 -9 9 % re d u c tio n ; sp o ru la tio n o f F. g ra m in e a ru m o n C . ro s e a -t re a te d st ra w : 9 1 -1 0 0 % re d u c tio n ; H ig h ly e ff e c tiv e fu n g a la n ta g o n is ts : C . ro s e a , F. e q u is e ti , C h a e to m iu m g lo b o s u m a n d E p ic o c c u s n ig ru m ; n o n -p a th o g e n ic F u s a ri u m sp p . e xh ib ite d m o d e ra te a n ta g o n is m ; Y e a st s w e re w e a k c o m p e tit o rs ; R e d u c tio n o f F u s a ri u m sp p . c o n id ia fo rm a tio n (m a iz e st u b b le b io a ss a y) : le ss e ff e c tiv e re d u c tio n in sp o ru la tio n th a n re p o rt e d fo r w h e a t st ra w ; st ro n g e st a n ta g o n is t: C . ro s e a ; F ie ld tr ia ls : M a iz e st a lk s: V a ria tio n in re su lts ; M o st c o n si st e n t re d u c tio n o f F u s a ri u m c o lo n iz a tio n b y C . ro s e a ; M a iz e e a rs : E a r tr e a tm e n ts w ith C . ro s e a a n d C la d o s p o ri u m c la d o s p o ri o id e s re d u c e d c o lo n iz a tio n o f ke rn e ls w ith b o th F. ve rt ic ill io id e s a n d F. g ra m in e a ru m (5 0 % re d u c tio n ); F. p ro lif e ra tu m c o lo n iz a tio n re d u c e d b y C . c la d o s p o ri o id e s a n d F. e q u is e ti A p p lic a tio n o f a n ta g o n is ts o n flo w e rin g m a iz e e a rs : p ro m is in g re su lts in p re lim in a ry fie ld tr ia ls ; fu rt h e r e xp e rim e n ts u n d e r d is e a se c o n d u c iv e c o n d iti o n s n e e d e d ; se ve ra la n ta g o n is ts e xh ib ite d p o te n tia lt o c o n tr o l F u s a ri u m sp p . in w h e a t a n d m a iz e c ro p re si d u e s p o st h a rv e st , a n d a t th e flo w e rin g e a r st a g e s L u o n g o e t a l., 2 0 0 5 (C o n ti n u e d )
T A B L E 1 | C o n ti n u e d B io c o n tr o l mi c ro o rg a n is m F u s a ri u m s p p . s tu d ie d Te s t s y s te m w it h d e ta il s o f e x p e ri me n ta l mo d e l R e d u c ti o n c ri te ri a A p p li c a ti o n R e fe re n c e (s ) M a iz e e a rs : p re -i n o c u la tio n o f si lk s w ith p o te n tia la n ta g o n is ts ; si lk o f ta g g e d e a rs a t th e b lo o m in g st a g e ; su b se q u e n t in o c u la tio n o f si lk s w ith F. ve rt ic ill io id e s , F. p ro lif e ra tu m a n d F. g ra m in e a ru m ; id e n tific a tio n o f F. ve rt ic ill io id e s , F. p ro lif e ra tu m a n d F. g ra m in e a ru m : c o lo n y m o rp h o lo g y a n d m ic ro sc o p ic e xa m in a tio n L a c tic a c id b a c te ria l is o la te s fr o m m a iz e tis su e s c o lle c te d in m a iz e fie ld s (6 6 is o la te s) ; P e d io c o c c u s p e n to s a c e u s L 0 0 6 A va rie ty o f F. ve rt ic ill io id e s a n d F. p ro lif e ra tu m st ra in s fr o m th e IN R A M yc S A c o lle c tio n P o te n tia lo f P. p e n to s a c e u s (L 0 0 6 ) is o la te d fr o m m a iz e le a f to su p p re ss fu m o n is in -p ro d u c in g fu n g a lg ro w th . In vi tr o : A n tif u n g a la c tiv ity a g a in st F. ve rt ic ill io id e s a n d F. p ro lif e ra tu m : O ve rla y M R S a g a r p la te m e th o d ; se le c tio n o f th e m o st e ffic ie n t is o la te ; Id e n tific a tio n o f th e m o st e ffic ie n t a n tif u n g a ll a c tic a c id b a c te ria li so la te : b io c h e m ic a la n d p h ys io lo g ic a l c h a ra c te riz a tio n (A P I 5 0 C H L te st ); 1 6 S rR N A g e n e se q u e n c in g ; A n tif u n g a ls p e c tr u m o f P. p e n to s a c e u s L 0 0 6 o n so lid m e d iu m : P. p e n to s a c e u s L 0 0 6 te st e d a g a in st a ra n g e o f F. ve rt ic ill io id e s a n d F. p ro lif e ra tu m st ra in s; O ve rla y M R S a g a r p la te m e th o d ; P ro d u c tio n o f a c tiv e a n tif u n g a lm e ta b o lit e s b y P. p e n to s a c e u s L 0 0 6 : c u lti va tio n in M R S b ro th ; sa m p lin g d u rin g 1 2 0 h in c u b a tio n p e rio d ; m e a su re m e n t o f p H , c e ll g ro w th a n d a n tif u n g a la c tiv ity o f c e ll-fr e e su p e rn a ta n t; d e te rm in a tio n o f a n tif u n g a la c tiv ity ; C h a ra c te riz a tio n o f P. p e n to s a c e u s L 0 0 6 c e ll-fr e e c u ltu re su p e rn a ta n t: d e te rm in a tio n o f h e a t st a b ili ty a n d th e e ff e c ts o f p H a n d p ro te o ly tic e n zy m e tr e a tm e n ts o n a n tif u n g a la c tiv ity In vi tr o : A n tif u n g a la c tiv ity a g a in st F. ve rt ic ill io id e s a n d F. p ro lif e ra tu m : 8 9 % o f la c tic a c id b a c te ria li so la te s w e re a b le to in h ib it fu n g a lg ro w th ; a n tif u n g a la c tiv ity m a xi m a lt o w a rd th e e n d o f th e e xp o n e n tia lp h a se o f g ro w th ; Id e n tific a tio n o f th e m o st e ffic ie n t a n tif u n g a ll a c tic a c id b a c te ria li so la te : P. p e n to s a c e u s L 0 0 6 (1 0 0 % se q u e n c e si m ila rit y w ith P. p e n to s a c e u s A T C C 2 5 7 4 5 ); A n tif u n g a ls p e c tr u m o f P. p e n to s a c e u s L 0 0 6 o n so lid m e d iu m : P. p e n to s a c e u s L 0 0 6 in h ib ite d th e g ro w th o f a ll fu n g a ls tr a in s te st e d ; P ro d u c tio n o f a c tiv e a n tif u n g a lm e ta b o lit e s b y P. p e n to s a c e u s L 0 0 6 : a n tif u n g a la c tiv ity in c re a se d w ith in c u b a tio n tim e ; a n tif u n g a ls u b st a n c e s a re p o ss ib ly se c o n d a ry m e ta b o lit e s; p H d e c re a se d (p H 6 .5 to 3 .8 ) d u rin g in c u b a tio n ; C h a ra c te riz a tio n o f P. p e n to s a c e u s L 0 0 6 c e ll-fr e e c u ltu re su p e rn a ta n t: a n tif u n g a la c tiv ity w a s n o t re d u c e d b y h e a t a n d p ro te o ly tic e n zy m e tr e a tm e n ts ; a n tif u n g a lc o m p o u n d s n o t p ro te in a c e o u s; a n tif u n g a l a c tiv ity lo st a t p H 7 ; a n tif u n g a la c tiv ity w a s a sc rib e d to th e p re se n c e o f o rg a n ic a c id s, e xc lu d in g la c tic a c id A p p lic a tio n o f P. p e n to s a c e u s L 0 0 6 c a n p o ss ib ly im p ro ve si la g e q u a lit y; re su lts o b ta in e d in vi tr o n e e d to b e e xt e n d e d to in p la n ta st u d ie s a n d fie ld tr ia ls D a lie e t a l., 2 0 1 0 (C o n ti n u e d )
T A B L E 1 | C o n ti n u e d B io c o n tr o l mi c ro o rg a n is m F u s a ri u m s p p . s tu d ie d Te s t s y s te m w it h d e ta il s o f e x p e ri me n ta l mo d e l R e d u c ti o n c ri te ri a A p p li c a ti o n R e fe re n c e (s ) B a c ill u s m o ja ve n s is st ra in s R C C 1 0 1 (A T C C 5 5 7 3 2 ) (p a te n te d ); N R R L B 1 4 6 9 9 ; N R R L B 1 4 7 0 1 ; N R R L B 1 4 7 0 3 to N R R L B 1 4 7 0 6 ; N R R L B 1 4 7 0 8 to N R R L B 1 4 7 1 2 ; B . m o ja ve n s is rif a m p in m u ta n t R R C 1 1 2 rif ; B . m o ja ve n s is fu sa ric a c id re si st a n t m u ta n t R R C 1 1 2 fa F. ve rt ic ill io id e s st ra in s: M R C 8 2 6 (s ym p to m le ss e n d o p h yt ic st ra in ), “P a tg u s” (v iru le n t st ra in ), 4 0 8 (v iru le n t w ild ty p e st ra in ), a n d U V 2 8 (n o n -f u sa ric a c id p ro d u c in g m u ta n t st ra in ) In p la n ta re d u c tio n o f m a iz e se e d lin g st a lk le si o n s b y th e b a c te ria le n d o p h yt e B . m o ja ve n s is . In p la n ta : B . m o ja ve n s is (s tr a in s R C C 1 0 1 ; N R R L B 1 4 6 9 9 ; N R R L B 1 4 7 0 1 ; N R R L B 1 4 7 0 3 to N R R L B 1 4 7 0 6 ; N R R L B 1 4 7 0 8 to N R R L B 1 4 7 1 2 ) in o c u la te d Z e a m a ys “E a rly S u n g lo w ” se e d s w e re c u lti va te d fo r 3 5 d a ys in a p la n t g ro w th lig h t ro o m a n d in o c u la te d w ith a sp o re su sp e n si o n o f F. ve rt ic ill io id e s “P a tg u s” ; B . m o ja ve n s is R R C 1 1 2 fa in o c u la te d Z e a m a ys “P io n e e r 3 1 4 0 ” se e d s w e re c u lti va te d a n d in o c u la te d a s d e sc rib e d a b o ve w ith F. ve rt ic ill io id e s st ra in s M R C 8 2 6 , 4 0 8 a n d U V 2 8 ; B . m o ja ve n s is R R C 1 1 2 fa in o c u la te d Z e a m a ys “E a rly S u n g lo w ” se e d s w e re c u lti va te d a s d e sc rib e d a b o ve a n d in o c u la te d w ith F. ve rt ic ill io id e s st ra in s “P a tg u s” a n d U V 2 8 ; D e te rm in a tio n o f st a lk le si o n d e ve lo p m e n t; M e a su re m e n t o f th e le n g th o f n e c ro tic le si o n s a n d st a lk d ia m e te rs In p la n ta : R a n g e o f B . m o ja ve n s is st ra in s + F. ve rt ic ill o id e s “P a tg u s” (“ E a rly S u n g lo w ” m a iz e ): 2 4 -5 8 % re d u c tio n in st a lk le si o n le n g th ; la rg e d iff e re n c e s in th e a b ili ty to re d u c e le si o n s; B . m o ja ve n s is R C C 1 0 1 e xh ib ite d 5 8 % re d u c tio n ; B . m o ja ve n s is R R C 1 1 2 fa + F. ve rt ic ill io id e s st ra in s M R C 8 2 6 , 4 0 8 a n d U V 2 8 (“ P io n e e r 3 1 4 0 ” m a iz e ): 3 0 -4 1 % re d u c tio n in st a lk le si o n le n g th ; B . m o ja ve n s is R R C 1 1 2 fa si g n ific a n tly (P = 0 .0 5 ) re d u c e d st a lk le si o n le n g th s c a u se d b y F. ve rt ic ill io id e s “P a tg u s” o n “E a rly S u n g lo w ” m a iz e (5 4 % re d u c tio n ); B . m o ja ve n s is R R C 1 1 2 fa : 7 0 % re d u c tio n in st a lk le si o n le n g th ; re d u c tio n n o t si g n ific a n tly d iff e re n t fr o m re su lts fo r th e R R C 1 0 1 w ild ty p e st ra in a n d rif a m p in m u ta n t st ra in (R R C 1 1 2 rif ); S ig n ific a n t (P ≤ 0 .0 5 ) re d u c tio n in st a lk le si o n le n g th b y th e b a c te riu m re g a rd le ss o f its a b ili ty to to le ra te fu sa ric a c id ; F. ve rt ic ill o id e s U V 2 8 si g n ific a n tly (P ≤ 0 .0 5 ) re d u c e d m a iz e st a lk d ia m e te r; n o e n h a n c e d e ff e c t w h e n th e fu n g u s w a s c o -i n o c u la te d w ith B . m o ja ve n s is A p p lic a tio n o f B . m o ja ve n s is fo r su p p re ss io n o f se e d lin g d is e a se in m a iz e : to p ro o f th e e ffic a c y o f B . m o ja ve n s is a s b io c o n tr o l a g e n t a d d iti o n a ls tu d ie s sh o u ld b e p e rf o rm e d in vi tr o a n d in th e fie ld u til iz in g m u ta n ts a n d w ild -t yp e st ra in s o f b a c te ria a n d n o n -f u m o n is in p ro d u c in g fu n g i; m o re p a th o lo g ic a lf a c to rs sh o u ld a ls o b e e va lu a te d B a c o n a n d H in to n , 2 0 1 1 F B1 , F u m o n is in B1 ; C F U , C o lo n y fo rm in g u n its ; P D A , P ota to de xtr os e a ga r; MR S br oth /a ga r, de Ma n , R ogos a a n d S h a rpe br oth /a ga r.
(
Luongo et al., 2005
). C. rosae exhibited potential to control
Fusarium spp. in maize at the flowering ear stages and in crop
residues post-harvest. Food-grade yeasts are also considered
ideal biocontrol microorganisms, as they are generally genetically
stable, effective at low concentrations, easy to cultivate,
capable to survive under adverse environmental conditions,
compatible with commercial processing, and resistant to
pesticides.
Trichoderma
spp.
Trichoderma spp. are considered effective biocontrol agents
because of their repertoire of extracellular lytic enzymes that
cause necrotrophic action through lysis of fungal cell walls
as well as the role they play in ISR in plants (
Bacon et al.,
2001; Hermosa et al., 2012
). Trichoderma mainly colonizes the
rhizosphere and intercellular root areas of plants, and maintains
interactions by promoting plant growth and providing protection
against infections, while utilizing plant sucrose to facilitate root
colonization (
Hermosa et al., 2012
). Plant disease severity is
reduced in the presence of Trichoderma by inhibition of a wide
range of plant pathogens through antagonistic and mycoparasitic
action; ISR or induction of localized resistance. Trichoderma
is also able to withstand toxic metabolites that are produced
by the plant in response to invasion. Plants are able to detect
pathogen- or microbe associated molecular patterns (MAMPs),
which leads to activation of defense mechanisms and eventually
synthesis of antimicrobial compounds. Certain Trichoderma
strains produce a variety of MAMPs, contributing to activation of
plant defense responses. Salicylic acid, jasponic acid and ethylene
play a key role in plant immunity and hormone-signaling
pathways as well as defense response pathways of the hormones
abscisic acid, indole-3-acetic acid, and gibberellin (
Pieterse
et al., 2009
). Indole-3-acetic acid produced by Trichoderma
contributes to ethylene biosynthesis, which in turn stimulates
abscisic acid biosynthesis. Depending on Trichoderma stimuli,
phytohormone homeostasis will control plant development and
immune responses. Trichoderma chitinases also release fungal
chitin oligosaccharides, and elicit ISR by jasmonic acid/ethylene
dependent pathways, thereby triggering defense responses in
plants. A polyketide synthase/non-ribosomal peptide synthetase
hybrid enzyme of Trichoderma virens is involved in plant
interactions and was shown to induce plant defense responses
(
Mukherjee et al., 2012
). Several Trichoderma spp. with
GRAS status, including Trichoderma viride and Trichoderma
harzianum, are capable of effectively reducing F. verticillioides
(= F. moniliforme) growth and fumonisin production in
vitro and in planta (
Calistru et al., 1997; Larkin and Fravel,
1998; Yates et al., 1999
; Table 1). The inhibitory effect on
F. verticillioides growth when co-cultured with Trichoderma
spp. can be attributed to antibiosis through production of
volatile compounds, extracellular enzymes and antibiotics. The
antagonistic fungal species T. viride is widely used in
bio-fertilizers for biological control of soil borne plant-pathogenic
fungi in crops.
Non-Pathogenic Biocontrol Strains
Non-pathogenic strains of pathogenic species are often applied
for biocontrol (
Liu et al., 2013
). In this regard, moderate
suppression of toxigenic F. verticillioides and F. proliferatum
strains by non-pathogenic Fusarium strains was demonstrated by
Luongo et al.
(
2005
; Table 1).
The development of Fusarium biocontrol strains with reduced
mycotoxin production ability through RNA silencing technology
may be a useful tool for reducing mycotoxin contamination in
agricultural products (
McDonald et al., 2005
). Transformation of
F. graminearum with inverted repeat transgenes (IRT) containing
sequences of mycotoxin-specific regulatory genes results in
suppression of mycotoxin production. Other gene silencing
techniques involving deletion of ZFR1 of F. verticillioides, which
regulates sugar transporter genes and in turn affect fumonisin
biosynthesis during kernel colonization, resulted in significantly
less growth on maize kernel endosperm tissue (
Bluhm et al.,
2008
).
Rhizobacteria
Fusarium verticillioides is the most prevalent Fusarium spp.
present in the rhizoplane and endorhizosphere areas of maize,
while Arthrobacteria and Azotobacter are the predominant
bacterial genera (
Cavaglieri et al., 2005a
). Pathogens germinate
and colonize roots within a few days of planting, while biocontrol
rhizobacteria could be metabolically active during this period.
A number of rhizobacterial isolates of maize plants sampled
from a commercial maize field and exhibiting high NOIs with
F. verticillioides, including Arthrobacter globiformis, Azotobacter
armeniacus, Pseudomonas solanacearum, B. subtilis, Enterobacter
cloacae, and Microbacterium eoleovorans exhibited antifungal
activity in vitro by effectively reducing F. verticillioides growth
and FB
1production on maize meal extract agar (
Cavaglieri
et al., 2004, 2005a,b,c
) (Table 2). Maize seeds pre-treated with A.
armeniacus RC2, A. globiformis RC5, E. cloacae, M. eoleovorans,
and Bacillus sp. CE1 and evaluated in planta, resulted in
effective reduction of F. verticillioides growth in the rhizoplane
and endorhizosphere areas. A good correlation was observed
between results obtained from in vitro and in planta studies
(
Cavaglieri et al., 2005c
). Enterobacter cloacae exhibited potential
for biocontrol of root colonization by F. verticillioides. Inducible
Type 1 fimbrae of E. cloacae may play a role in the colonization
of roots (
Hinton and Bacon, 1995
). Rhizobacterial strains could
have potential application as seed inoculants to reduce F.
verticillioides colonization on root level, in the rhizoplane and
endorhizosphere areas (
Cavaglieri et al., 2005c
). Effectiveness of
a biocontrol organism to colonize the rhizosphere and its value as
biocontrol agent could, however, be influenced by environmental
conditions and the initial cell concentrations of the biocontrol
organism and the pathogen.
Antioxidants, Phenolic Compounds, and
Essential Oils
Several natural phenolic compounds derived from plants
are strong antioxidants and exhibit antimicrobial activity by
inhibiting the activity of key fungal enzymes, and are applied as
preservatives in the cosmetic, food and drug industries (Table 3).
These compounds are also considered promising antifungal
agents for controlling fungal growth and associated mycotoxin
production in agricultural crops pre-harvest, post-harvest, and
during storage.
T A B L E 2 | C u rr e n t in fo rma ti o n o n re d u c ti o n o f F u s a ri u m v e rt ic il li o id e s g ro w th a n d fu mo n is in B1 p ro d u c ti o n b y rh iz o b a c te ri a in v it ro a n d in p la n ta . R h iz o b a c te ri a l mi c ro o rg a n is m F u s a ri u m s p . s tu d ie d W a te r a c ti v it y (aw ) Te s t s y s te m w it h d e ta il s o f e x p e ri me n ta l mo d e l R e d u c ti o n c ri te ri a A p p li c a ti o n R e fe re n c e (s ) R h iz o b a c te ria li so la te s fr o m m a iz e p la n ts in It a ly : E n te ro b a c te r c lo a c a e F. ve rt ic ill io id e s (= F. m o n ili fo rm e ): Is o la te s fr o m m a iz e : M R C 8 2 6 , R R C 3 7 4 , R R C 4 0 8 , Is o la te s fr o m ric e : R R C 4 1 0 N /A E . c lo a c a e is a n e n d o p h yt ic sy m b io n t o f c o rn . R e d u c tio n o f F. ve rt ic ill io id e s ro o t c o lo n iz a tio n o f m a iz e se e d lin g s b y E . c lo a c a e : In p la n ta : D is tr ib u tio n o f E . c lo a c a e ro o t c o lo n iz a tio n : st e ril e m a iz e se e d in o c u la te d w ith E . c lo a c a e a n d c u ltu re d in tu b e s w ith so il; c u ltu re d in p la n t g ro w th ro o m s u n d e r lig h t; m ic ro sc o p ic e xa m in a tio n o f ro o t c o lo n iz a tio n ; In vi tr o : C u lti va tio n o f in o c u la te d se e d s o n P D A , d a m p fil te r p a p e r o r st e ril e so il; m ic ro sc o p ic e xa m in a tio n o f ro o t c o lo n iz a tio n ; D e te rm in a tio n o f a n ta g o n is m : c o -c u lti va tio n o n P D A ; e xa m in a tio n o f zo n e s o f in h ib iti o n ; m ic ro sc o p ic e xa m in a tio n o f se e d lin g ro o ts : lig h t m ic ro sc o p y; tr a n sm is si o n e le c tr o n m ic ro sc o p y; sc a n n in g e le c tr o n m ic ro sc o p y In p la n ta : E . c lo a c a e ro o t c o lo n iz a tio n : E . c lo a c a e b io lo g ic a lly a ss o c ia te d w ith m a iz e se e d lin g ro o ts ; o b se rv e d in te rn a lly a n d in th e rh iz o p la n e a re a s; o n m a iz e se e d lin g s E . c lo a c a e w a s d is tr ib u te d o ve r th e e p id e rm is a n d in te rn a lly in se ve ra ll o c a tio n s o f th e c o rt e x; n o b a c te ria o b se rv e d in th e e n d o d e rm is , b u t in te rc e llu la r w ith in th e o u te r m a rg in o f th e p e ric yc le ; E . c lo a c a e n o t o b se rv e d in th e p ith a re a ; p re se n t in st e m s a n d le a ve s; E . c lo a c a e d is tr ib u te d e xt e rn a lly a lo n g th e se c o n d a ry a n d p rim a ry se e d lin g ro o ts a s w e ll a s th e ro o t c a p o f th e p rim a ry ro o t; a m a tr ix -l ik e c a p su le o b se rv e d su rr o u n d in g th e b a c te ria lc e lls o n th e e xt e rn a l su rf a c e o f th e p rim a ry ro o t; E . c lo a c a e : n o d a m a g e to h o st c e lls ; n o re d u c tio n in p e rc e n ta g e g e rm in a tio n o r tim e o f g e rm in a tio n ; D e te rm in a tio n o f a n ta g o n is m : a ll b a c te ria li so la te s in h ib ite d g ro w th o f F. ve rt ic ill io id e s st ra in s E . c lo a c a e e xh ib ite d p o te n tia lf o r b io c o n tr o lo f ro o t c o lo n iz a tio n b y F. ve rt ic ill io id e s ; th e e n d o p h yt ic a ss o c ia tio n o f E . c lo a c a e w ith m a iz e e n h a n c e s its p o te n tia la s b io c o n tr o la g e n t H in to n a n d B a c o n , 1 9 9 5 R h iz o b a c te ria li so la te s fr o m m a iz e ro o ts , sa m p le d fr o m a c o m m e rc ia lm a iz e fie ld : A rt h ro b a c te r g lo b ifo rm is , A zo to b a c te r a rm e n ia c u s , P s e u d o m o n a s s o la n a c e a ru m , B . s u b ti lis F. ve rt ic ill io id e s is o la te s fr o m m a iz e ro o ts sa m p le d fr o m a c o m m e rc ia lm a iz e fie ld 0 .9 3 7 ; 0 .9 5 5 ; 0 .9 8 2 S c re e n in g p ro c e d u re s fo r se le c tin g rh iz o b a c te ria ls tr a in s w ith b io c o n tr o l e ff e c ts u p o n F. ve rt ic ill io id e s g ro w th a n d F B1 p ro d u c tio n : In vi tr o : D e te rm in a tio n o f N O Is : u til iz a tio n o f 1 7 c o m p o u n d s in m a iz e a s so le c a rb o n so u rc e ; se le c tio n o f is o la te s w ith th e h ig h e st N O Is ; A n tib io si s a n d a n tif u n g a la c tiv ity o f se le c te d is o la te s: 2 % M M E A ; a d ju st m e n t o f aw le ve ls ; in o c u la tio n a n d in c u b a tio n ; m e a su re m e n t o f zo n e s o f in h ib iti o n a n d c o lo n y d ia m e te rs ; F B1 le ve ls in M M E A c u ltu re s: H P L C a n a ly se s In vi tr o : D e te rm in a tio n o f N O Is : in fo rm a tio n o n e c o lo g ic a l si m ila rit y a n d c o e xi st e n c e w ith F. ve rt ic ill io id e s ; p e rc e n ta g e is o la te s a b le to u til iz e a ll c a rb o n so u rc e s: aw 0 .9 3 7 (5 8 % ), 0 .9 5 5 (2 0 % ) 0 .9 8 2 (7 5 % ); m o st c o m p e te n t st ra in s: A rt h ro b a c te r st ra in s a t a ll aw le ve ls ; A . a rm e n ia c u s , P. s o la n a c e a ru m a n d B . s u b ti lis c o m p e te n t a t aw 0 .9 5 5 a n d 0 .9 3 7 ; A n tib io si s a n d a n tif u n g a la c tiv ity o f se le c te d is o la te s: a ll b a c te ria li so la te s e ff e c tiv e ly in h ib ite d F. ve rt ic ill io id e s g ro w th ; m o st e ff e c tiv e g ro w th in h ib iti o n (P < 0 .0 0 1 ): A . g lo b ifo rm is a n d B . s u b ti lis is o la te s; a ll is o la te s si g n ific a n tly (P < 0 .0 0 1 ) re d u c e d th e g ro w th ra te a n d in c re a se d th e la g p h a se o f fu n g a lg ro w th ; B . s u b ti lis st ra in s e xh ib ite d th e st ro n g e st e ff e c ts ; F B1 le ve ls in M M E A c u ltu re s: re d u c e d F B1 le ve ls e xh ib ite d a t a ll aw le ve ls e va lu a te d ; P. s o la n a c e a ru m a n d B . s u b ti lis : 7 0 -1 0 0 % re d u c tio n a t a ll aw le ve ls ; A . a rm e n ia c u s : 6 5 % re d u c tio n a t aw 0 .9 5 5 A . a rm e n ia c u s R C 2 a n d R C 3 ; B . s u b ti lis R C 8 , R C 9 a n d R C 1 1 ; P. s o la n a c e a ru m R C 7 a n d R C 1 0 c o u ld h a ve va lu e fo r c o n tr o lo f F. ve rt ic ill o id e s ro o t c o lo n iz a tio n C a va g lie ri e t a l., 2 0 0 4 (C o n ti n u e d )
T A B L E 2 | C o n ti n u e d R h iz o b a c te ri a l mi c ro o rg a n is m F u s a ri u m s p . s tu d ie d W a te r a c ti v it y (aw ) Te s t s y s te m w it h d e ta il s o f e x p e ri me n ta l mo d e l R e d u c ti o n c ri te ri a A p p li c a ti o n R e fe re n c e (s ) P re d o m in a n t b a c te ria l is o la te s c o lo n iz in g th e m a iz e e n d o rh iz o sp h e re a n d is o la te d fr o m m a iz e ro o ts , sa m p le d fr o m a c o m m e rc ia lm a iz e fie ld : A . g lo b ifo rm is , A . a rm e n ia c u s To xi g e n ic F. ve rt ic ill io id e s m a iz e e n d o rh iz o sp h e re is o la te s fr o m m a iz e ro o ts , sa m p le d fr o m a c o m m e rc ia l m a iz e fie ld 0 .9 3 7 ; 0 .9 5 5 ; 0 .9 8 2 R h iz o b a c te ria a n d th e ir p o te n tia lt o c o n tr o lF . ve rt ic ill io id e s : e ff e c t o f m a iz e b a c te riz a tio n a n d in o c u lu m d e n si ty : In vi tr o : F. ve rt ic ill io id e s is o la te s p a ire d w ith e a c h b a c te ria ls tr a in in d u a lc u ltu re ; A n tib io si s a n d e ff e c t o n fu n g a lg ro w th ra te : M M E A ; in o c u la tio n a n d in c u b a tio n ; m e a su re m e n t o f zo n e s o f in h ib iti o n ; m e a su re m e n t o f c o lo n y d ia m e te rs ; F B1 le ve ls in M M E A c u ltu re s: H P L C a n a ly se s G re e n h o u se st u d ie s: E ff e c t o f se p a ra te a n d c o m b in e d b a c te ria lt re a tm e n ts o n F. ve rt ic ill io id e s ro o t c o lo n iz a tio n in th e rh iz o p la n e a n d e n d o rh iz o sp h e re a re a s; in o c u la tio n o f se e d s w ith rh iz o b a c te ria ls tr a in s; m o d ifie d tu b e a ss a y; d e te rm in a tio n o f F. ve rt ic ill io id e s C F U c o u n ts in th e rh iz o p la n e a n d e n d o rh iz o sp h e re a re a s In vi tr o : A n tib io si s a n d e ff e c t o n fu n g a lg ro w th ra te : e ff e c tiv e in h ib iti o n o f fu n g a lg ro w th a t aw 0 .9 5 5 a n d 0 .9 8 2 ; A . a rm e n ia c u s R C 2 a n d R C 3 in h ib ite d fu n g a l g ro w th o f 6 0 -1 0 0 % F. ve rt ic ill io d e s st ra in s a t aw 0 .9 5 5 –0 .9 8 2 ; A . g lo b ifo rm is R C 4 a n d R C 5 in h ib ite d fu n g a lg ro w th o f 6 9 -8 0 % o f F. ve rt ic ill io d e s s tr a in s a t aw 0 .9 5 5 -0 .9 8 2 ; A . a rm e n ia c u s R C 2 re d u c e d (5 6 -7 5 % ) F B1 a c c u m u la tio n a t aw 0 .9 5 5 ; A . g lo b ifo rm is R C 4 a n d R C 5 re d u c e d (2 0 -9 6 % ) F B1 a c c u m u la tio n a t aw 0 .9 5 5 a n d 0 .9 8 2 ; G re e n h o u se st u d ie s: S e e d s tr e a te d w ith A . a rm e n ia c u s R C 2 a n d A . g lo b ifo rm is R C 5 : 1 0 0 % in h ib iti o n o f fu n g a lg ro w th in th e rh iz o p la n e a n d e n d o rh iz o sp h e re a re a s; b a c te ria lm ix tu re tr e a tm e n t re su lte d in 1 0 0 % in h ib iti o n o f fu n g a lg ro w th in th e e n d o rh iz o sp h e re a re a A . a rm e n ia c u s R C 2 e xh ib ite d p o te n tia la s m a iz e se e d in o c u la n t fo r re d u c tio n o f F. ve rt ic ill io id e s ro o t c o lo n iz a tio n C a va g lie ri e t a l., 2 0 0 5 a E n d o rh iz o sp h e re b a c te ria li so la te s fr o m m a iz e ro o ts , sa m p le d fr o m a c o m m e rc ia l m a iz e fie ld : B a c te ria lm ix tu re 1 : E . c lo a c a e , M ic ro b a c te ri u m e o le o vo ra n s B a c te ria lm ix tu re 2 : P. s o la n a c e a ru m , B . s u b ti lis To xi g e n ic F. ve rt ic ill io id e s m a iz e e n d o rh iz o sp h e re is o la te s fr o m m a iz e ro o ts , sa m p le d fr o m a c o m m e rc ia l m a iz e fie ld 0 .9 3 7 ; 0 .9 5 5 ; 0 .9 8 2 In vi tr o in flu e n c e o f b a c te ria lm ix tu re s o n F. ve rt ic ill io id e s g ro w th a n d F B1 p ro d u c tio n : e ff e c t o f se e d s tr e a tm e n t o n m a iz e ro o t c o lo n iz a tio n : In vi tr o : A n tib io si s: M M E A ; a d ju st m e n t o f aw le ve ls ; F. ve rt ic ill io id e s is o la te s p a ire d w ith e a c h b a c te ria lm ix tu re in d u a l c u ltu re ; d iff e re n t b a c te ria li n o c u lu m si ze s e va lu a te d (1 0 8, 1 0 9a n d 1 0 1 0 c e lls /m l); m e a su re m e n t o f zo n e s o f in h ib iti o n ; A n tif u n g a la c tiv ity : M M E A ; a d ju st m e n t o f aw le ve ls ; p o u r-p la te m e th o d ; in o c u la tio n w ith F. ve rt ic ill io id e s is o la te s; m e a su re m e n t o f c o lo n y d ia m e te rs ; F B1 le ve ls in M M E A c u ltu re s: H P L C a n a ly se s; G re e n h o u se st u d ie s: E ff e c t o f c o m b in e d b a c te ria ls e e d tr e a tm e n ts o n F. ve rt ic ill io id e s ro o t c o lo n iz a tio n in th e rh iz o p la n e a n d In vi tr o : B a c te ria lm ix tu re 1 : A n tib io si s: fu n g a lg ro w th si g n ific a n tly (P < 0 .0 5 ) re d u c e d a t a ll aw le ve ls a n d in o c u lu m si ze s; in o c u lu m si ze 1 0 8c e lls /m le xh ib ite d th e st ro n g e st e ff e c t; A n tif u n g a la c tiv ity : si g n ific a n t (P < 0 .0 5 ) d e c re a se in fu n g a lg ro w th ra te a t aw 0 .9 5 5 a n d 0 .9 3 7 w ith a ll in o c u lu m si ze s; re d u c tio n in fu n g a lg ro w th ra te o b ta in e d a t aw 0 .9 8 2 w ith 1 0 9a n d 1 0 1 0c e lls /m l; F B1 p ro d u c tio n : o n ly re d u c e d a t aw 0 .9 5 5 b y a ll in o c u lu m si ze s; B a c te ria lm ix tu re 2 : A n tib io si s: fu n g a lg ro w th m o st e ff e c tiv e ly (P < 0 .0 5 ) re d u c e d a t aw 0 .9 3 7 w ith 1 0 8a n d 1 0 9c e lls /m l; A n tif u n g a la c tiv ity : si g n ific a n t (P < 0 .0 5 ) re d u c tio n in fu n g a lg ro w th ra te o b ta in e d a t aw 0 .9 8 2 w ith 1 0 1 0 c e lls /m la n d a t aw 0 .9 5 5 w ith 1 0 9c e lls /m l; n o e ff e c t w ith 1 0 8c e lls /m l; F B1 p ro d u c tio n n o t re d u c e d b y a n y o f th e in o c u lu m si ze s; E . c lo a c a e a n d M . o le o vo ra n s e xh ib ite d p o te n tia la s m a iz e se e d in o c u la n ts fo r re d u c tio n o f F. ve rt ic ill io id e s ro o t c o lo n iz a tio n , i.e . p re ve n tio n o f ve rt ic a l tr a n sm is si o n o f F. ve rt ic ill io id e s C a va g lie ri e t a l., 2 0 0 5 b (C o n ti n u e d )
