Microbes and Microbial Products as Herbicides - ACS Publications

Chapter 17

Bioherbicide Technology A n Industrial Perspective James S. Bannon , James C. White, D. Long, J. A. Riley, J . Baragona, M . Atkins, and R. H. Crowley

Downloaded by UNIV OF MICHIGAN ANN ARBOR on December 16, 2017 | http://pubs.acs.org Publication Date: September 25, 1990 | doi: 10.1021/bk-1990-0439.ch017

1

Mycogen Corporation, 3303 McDonald Avenue, Ruston, LA 71270

Technology which supports commercial development of new bioherbicides will continue to require integrated, mul tidisciplinary research efforts from public/private sectors for optimization of success. Discov ery/isolation of stable, virulent bioherbicide candi dates as well as strain improvement/long-term preserva tion form the foundation for commercial bioherbicide development. An economical, profitable means of propagule production is essential in determination of fermentation parameters, time of propagule harvest, and subsequent processing. Processing, which includes stabilization and formulation, is based on biological requirements of the organism for maintenance in a viable state prior to marketing and subsequent transi tion to a virulent state upon delivery to the weedy host. Upon delivery to the host, a major barrier to consistent performance of foliar-applied bioherbicides is the requirement for free moisture. Development of technology to circumvent this requirement will be tantamount to the successful commercialization of certain foliar-applied bioherbicides. Natural antagonists have continued to suppress most pests including weeds f o r thousands of years (U . Weed predators such as insects and f i s h have accounted f o r s u c c e s s f u l b i o l o g i c a l c o n t r o l of p r i c k l y pear (Opuntia sp.), Klamath weed (Hypericum perforatum L.), and c e r t a i n aquatic species (2-4). The use of perpetuating insect and f i s h species as well as obligate plant p a r a s i t i c rusts (5,6) has been defined as the c l a s s i c a l b i o l o g i c a l c o n t r o l t a c t i c (2) . I t i s i n t e r e s t i n g to note the documentation of the c l a s s i c a l t a c t i c to control Canada t h i s t l e [Cirsium arvense (L.) Scop.] i n 1894 (&) . Although the e f f i c a c y of the c l a s s i c a l approach has been demon s t r a t e d , there i s l i t t l e or no p r o f i t i n c e n t i v e f o r industry. Therefore, research and development of t h i s type of b i o c o n t r o l strategy w i l l be conducted primarily by the public sector. 1

Current address: Ε. V . Smith Research Center, Alabama Agricultural Experiment Station, Route 1, Box 138, Shorter, A L 36075 0097-6156/90A)439-O305$06.00/O © 1990 American Chemical Society

Hoagland; Microbes and Microbial Products as Herbicides ACS Symposium Series; American Chemical Society: Washington, DC, 1990.


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The bioherbicide approach i s the other b i o c o n t r o l t a c t i c , as defined i n several reviews (9-11). This approach provides the best opportunity f o r p r o f i t a b l e commercial bioherbicide development as exemplified by DeVine [Phytophthora palmivora (Butl.)] (12.) and Collego [Colletotrichum gloeosporioides (Penz.) Sacc. f. sp. aeschynomene] 113). DeVine i s an e f f i c a c i o u s bioherbicide for the c o n t r o l of s t r a n g l e r v i n e [Morenia odorata (H.&A.) L i n d l . ] i n citrus. Since DeVine i s e f f i c a c i o u s within the s o i l matrix and k i l l s seedling stranglervine plants before they become established, f r e e moisture, i . e . , d e w / i r r i g a t i o n , i s not a requirement to optimize a c t i v i t y of t h i s bioherbicide. Moisture found i n the s o i l matrix i s s u f f i c i e n t f o r s t r a n g l e r v i n e seed germination, which subsequently causes an increase i n P . palmivora l e v e l s , thereby c o n t r o l l i n g the weedy host ( 1 2 ) · However, free moisture enhances efficacy of C o l l e g o (13 1 4 ) and other foliar epiphytic bioherbicides (15-191 on t h e i r respective hosts. r

Free moisture i s the most important environmental component of the disease t r i a n g l e i . e . the i n t e r a c t i o n of the f o l i a r epiphytic bioherbicide with i t s host and the environment. More importantly, an understanding of host-pathogen physiology/biochemistry as i n f l u enced by free moisture and other environmental factors w i l l be more l i k e l y to y i e l d solutions to bioherbicide e f f i c a c y problems caused by i n s u f f i c i e n t free moisture following host i n o c u l a t i o n . Experiments described below examine host-pathogen physiology and i d e n t i f y f a c t o r s that would improve bioherbicide e f f i c a c y and performance consistency with focus on free moisture i n t e r a c t i o n . A d d i t i o n a l l y , technology required to discover, maintain, and produce b i o h e r b i cides f o r commercialization i s described below. Bioherbicide

Discovery

Since free moisture determines bioherbicide performance consistency and e f f i c a c y , i t i s expected that research and development s t r a t e gies would be guided by t h i s facet of the disease t r i a n g l e . Bowers (2JL) has reviewed the r o l e of industry i n discovery of hosts p e c i f i c bioherbicides. The bioherbicide industry must continue to depend on the expertise of p u b l i c sector s c i e n t i s t s i n diverse locations to discover h o s t - s p e c i f i c bioherbicides. It i s axiomatic that the requirement f o r free moisture w i l l vary among b i o h e r b i cides. Thus, an assessment of t h i s environmental v a r i a b l e should be foremost i n preliminary studies on bioherbicide e f f i c a c y . The a v a i l a b i l i t y of free moisture i n the target weed market and the response of the bioherbicide to free moisture are primary determinants i n the d e c i s i o n to commercialize respective candidates. For example, the lawn and t u r f market would be more l i k e l y to augment natural free moisture through frequent i r r i g a t i o n than c e r t a i n row crop markets. Thus, e f f i c a c y of e p i p h y t i c f o l i a r b i o h e r b i c i d e candidates may be enhanced by matching the bioherbicide candidate with the appropriate market. The survey method, which u t i l i z e s the weedy host to screen pathogens, has been u t i l i z e d to discover most of the h o s t - s p e c i f i c b i o h e r b i c i d e candidates c u r r e n t l y under development. Again, the disease t r i a n g l e , i . e . , the host, bioherbicide pathogen, and the environment determine bioherbicide e f f i c a c y . Often, disease symptoms may not occur i n epidemic proportions. As such, manifestation of disease symptoms may occur i n a very narrow time span when a l l factors of the disease t r i a n g l e i n t e r a c t . Thus, discovery of hosts p e c i f i c , f o l i a r , epiphytic bioherbicide candidates i s dependent on


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Bhhcrbicide Technology

307

astute observation when symptoms are manifested on the host. T r a d i t i o n a l l y , s c i e n t i s t s i n the public sector have discovered most bioherbicide candidates because of proximity to f i e l d experiments, research s t a t i o n s , and more importantly, f a m i l i a r i t y with plant diseases and symptomology. U t i l i z a t i o n of the survey method to discover bioherbicide candidates by the b i o p e s t i c i d e industry w i l l continue a l b e i t primarily through l i c e n s i n g e f f o r t s . An a l t e r n a t i v e approach to discovery of bioherbicide candidates by the survey method i s a genetic approach. In a recent review, Sands et al. (Chapter 1 0 , t h i s volume; Sands, D. C. et al. Weed T e c h n o l . i n press) have stated that t h i s approach increases the number of fungi useful as bioherbicides. Because broad host range pathogens are u t i l i z e d i n t h i s approach, s e l e c t i v e weed c o n t r o l must be achieved through mutation technology. Pathogen d e l i m i t a t i o n must occur to r e s t r i c t host range and prevent spread and s u r v i v a l of t h i s type of bioherbicide. Pathogen d e l i m i t a t i o n provides a method to c o n t r o l the amount of b i o h e r b i c i d e i n the environment. Successful examples of pathogen d e l i m i t a t i o n of bioherbicide candidates have recently been reported ( 2 1 ; M i l l e r , R.


r

V.

et

al.

Can.

J.

Microbiol.

r

in

press).

Successful discovery of e f f i c a c i o u s bioherbicides w i l l depend on implementation of an objective, balanced research strategy. Such a research strategy w i l l depend on a cooperative research e f f o r t between public and private sector s c i e n t i s t s . Focus on components of the disease t r i a n g l e and target market c h a r a c t e r i s t i c s w i l l be tantamount to successful discovery of bioherbicides. Bioherbicide

Culture

Maintenance

Maintenance of virulence and v i a b i l i t y of bioherbicide cultures i s a v i t a l component of any commercially s u c c e s s f u l research and development e f f o r t . Several reviews on s p e c i f i c technology related to preservation and long-term storage of microorganisms have been w r i t t e n (22-25) r and s p e c i f i c d e t a i l s w i l l be omitted from t h i s review. S e n s i t i v i t y of bioherbicide organisms to various preservation techniques w i l l vary according to the s p e c i f i c organism. Selection of the optimum technique to preserve v i a b i l i t y , v i r u l e n c e , and pathogenicity i s guided by the type of propagule being preserved, pathogen species, and numerous other factors. Alternaria cassiae J u r a i r and Khan i s being developed as a bioherbicide to control sicklepod (Cassia obtusifolia L.). S i c k l e pod plants are inoculated with conidia of t h i s bioherbicide. Thus, i t i s desirable to preserve v i a b i l i t y , virulence, and pathogenicity of A. cassiae conidia. Accelerated ageing techniques are used r o u t i n e l y to p r e d i c t v i a b i l i t y of seed l o t s (2JL) . Since fungal spores are s i m i l a r to plant seed i n c e r t a i n aspects, an accelerated ageing technique was developed to rapidly assess A. cassiae conidia preservation methods. A. cassiae conidia were l y o p h i l i z e d according to established methodology. Lyophilized conidia were sealed i n glass ampules and stored i n an oven at 4 0° C f o r various time periods p r i o r to bioassay on sicklepod p l a n t s . Non-lyophilized conidia were stored i n open containers i n the same oven at 40° C. Non-lyophilized conidia are normally stored i n a desiccator at 5 ° C, and c o n i d i a were maintained i n t h i s manner as a c o n t r o l . Results of t h i s study show that v i a b i l i t y and v i r u l e n c e of A. cassiae are preserved best when conidia are l y o p h i l i z e d (Figure 1 ) .


308



Thus, moisture removal i s c r i t i c a l to preserve the i n t e g r i t y of A. cassiae bioherbicide propagules. Maintenance o f stock cultures f o r fermentation and production o f bioherbicide candidates i s another important aspect of bioherbicide culture maintenance (22). Uniformity and s t a b i l i t y of stock c u l t u r e s subsequently determine homogeneity of b i o h e r b i c i d e propagules. For example, v i r u l e n c e of A. cassiae d e c l i n e s a f t e r four culture transfers (Figure 2.). Production of homogeneous b i o herbicide propagules i s dependent upon a uniform source of inoculum. Bioherbicide

Propagule

Production

Bioherbicide propagules may be spores, c e l l s , mycelia, s c l e r o t i a , chlamydospores, e t c . Propagules of the commercial bioherbicides, DeVine and Collego, are chlamydospores and spores, r e s p e c t i v e l y . Production of b i o h e r b i c i d e propagules has been reviewed recently (27,281, and i t i s beyond the purpose of t h i s report to elaborate the d e t a i l s of fermentation processes. Bioherbicide propagules may be produced by e i t h e r s o l i d state or submerged fermentation processes. Both Collego and DeVine are produced by submerged fermentation process. Although s o l i d state fermentations may approximate natural conditions f o r fungal growth (22.), submerged fermentations may be more economical. A. cassiae conidia are produced by s o l i d state fermentation (15). The fermentation process and subsequent processing determine the e f f i c a c y of a bioherbicide propagule. A thorough understanding of the e f f e c t s of fermentation and formulation on v i a b i l i t y and v i r u l e n c e i s required to guide these processes. For example, the carbon source i n the fermentation medium has a profound e f f e c t on Fusarium lateritium Nees ex. Fr. e f f i c a c y (Figure 3). A d d i t i o n a l l y , carbon source and other components of the medium and i t s physical/chemical environment determine i n d u c t i o n / y i e l d of bioherb i c i d e propagules. A f t e r an economical y i e l d l e v e l of bioherbicide propagule has been achieved i n a fermentation process, formulation becomes a c r i t i c a l f a c t o r which influences product e f f i c a c y . Because the fermentation must be stopped at a point when v i r u l e n c e / v i a b i l i t y are optimum, the l i v e b i o h e r b i c i d e propagule must be s t a b i l i z e d , formulated, and packaged. Physiology of dehydration and rehydrat i o n must be examined i f the s t a b i l i z a t i o n process i n v o l v e s propagule drying. I t has been stated that spore dehydration and rehydration research was the greatest challenge i n the development of Collego (11). The chlamydospore formulation of DeVine required a r e f r i g e r a t e d d i s t r i b u t i o n system to maintain s t a b i l i t y (12) . Host-Pathogen-Environment

Interactions

E f f e c t s of F r e e M o i s t u r e . A v a i l a b i l i t y of free moisture a f t e r host i n o c u l a t i o n with a f o l i a r , e p i p h y t i c b i o h e r b i c i d e i s c r i t i c a l to the i n i t i a t i o n of the i n f e c t i o n process. Experiments on free moisture (dew) interactions with other environmental parameters and the host-pathogen system a r e u s u a l l y conducted in temperaturec o n t r o l l e d dew chambers. This type chamber i s constructed such that the p l a n t r a d i a t e s e n e r g y to a heat sink which i s the temperature-controlled w a l l of the chamber. The humid a i r i n the chamber i s also temperature controlled, and dew forms on the plant



ΒΑΝΝΟΝ ET AL

Bioherbicide Technology

309

Weeks •15C

^ 4 0 C m 4 0 eu

Figure 1. E f f e c t of Alternaria cassiae spore storage environs on sicklepod mortality. A. cassiae spores stored i n a desicca t o r r e f r i g e r a t e d at 5° C was the control treatment. A sublot of these spores was stored s i m i l a r l y at 40° C i n sealed glass ampoules at 40° C a f t e r l y o p h i l i z a t i o n (L). Another sublot was stored i n open containers at 40° C but not l y o p h i l i z e d .

Number of Sequential Transfers Figure 2. E f f e c t of sequential, weekly culture t r a n s f e r on sicklepod control by Alternaria cassiae grown on V-8 agar (52). Spores were harvested from plates i n a solution of 0.02% (v/v) Sterox NJ surfactant. Sicklepod plants i n the cotyledon to 1l e a f growth stage were inoculated with A. cassiae spores (10 spores/ml), then subjected to a 6-h dew p e r i o d at 25° C. Sicklepod plants were evaluated 14 days a f t e r inoculation. 5



310


when the host plant temperature reaches or f a l l s below the dew point of the a i r . In the host establishment of plant pathogens for experimental purposes the incubation period frequently exceeds 24 h. In t h i s case the period of host t i s s u e wetness w i l l approximate the actual incubation time. I t i s d e s i r a b l e f o r a b i o h e r b i c i d e to i n f e c t plant t i s s u e with minimum dew requirements because (a) the period of host t i s s u e wetness w i l l vary considerably under natural f i e l d conditions, and (b) the unpredictable frequency of dew occurrence i n nature. I f the experimental dew p e r i o d i n a dew chamber i s b r i e f , the actual period of host t i s s u e wetness may not c o r r e l a t e with the actual incubation time. This may be dependent on host species as well as i n t e r a c t i o n of the host species with the physic a l parameters of the dew chamber. A lengthy dew period may be s u f f i c i e n t to induce runoff and displace propagules from the host. A lengthy dew period may also give misleading r e s u l t s with hostpathogen-chemical herbicide i n t e r a c t i o n s . Incubation of the host t i s s u e a f t e r i n o c u l a t i o n with a plant pathogen or bioherbicide i s the most important f a c t o r determining success (efficacy) of the i n o c u l a t i o n (i£L) . When v e l v e t l e a f i s inoculated with F. lateritium spores (10 spores/ml) harvested from p e t r i plates 131,32), incubation i n an environment conducive to dew formation i s required immediately a f t e r inoculation (Figure 4). A delay of 4 h from host i n o c u l a t i o n u n t i l the occurrence of dew resulted i n a 50% decline i n e f f i c a c y of t h i s bioherbicide. "Predisposition" i s a term used to describe the influence of environmental factors on host s u s c e p t i b i l i t y . Environmental pred i s p o s i t i o n has been reviewed e x t e n s i v e l y by Colhoun (JL3.) · Research has shown that the decline i n bioherbicide e f f i c a c y of F. lateritium caused by a delay i n occurrence of dew (Figure 4) can be circumvented by predisposing the host with r a i n . Predisposition of v e l v e t l e a f with simulated r a i n (1.3 cm) r e s u l t e d i n nearly 100% k i l l a f t e r a 1-h delay i n dew occurrence (Figure 5). 6

Effect

of Spore Dormancy. Spore dormancy may contribute to a decline i n bioherbicide e f f i c a c y . Spore dormancy i n fungal spores may be maintained by chemicals as well as metabolic blocks (J3A) . The presence of i n h i b i t o r s may be indicated by an increase i n e f f i cacy upon addition of an adsorbent such as a c t i v a t e d charcoal to the bioherbicide at the time of i n o c u l a t i o n . This was the case when adsorbent was added to F. lateritium spores (10 spores/ml) and the occurrence of dew (20 h) was delayed f o r 8 h a f t e r i n o c u l a t i o n (Figure 6) . The agent causing spore dormancy may be synthesized by e i t h e r the spore or host. 6

Effect

of Chemical H e r b i c i d e s . The i n t e r a c t i o n of chemical herbicides with bioherbicides has been reported as a means to improve the spectrum of weed control as well as e f f i c a c y of the b i o h e r b i cide (9 35). i n the laboratory evaluation of chemical h e r b i cide/bioherbicide combinations, i t i s important to study the e f f i cacy of each component separately p r i o r to evaluation of combinat i o n treatments. Thus, the a c t i v i t y of a given chemical herbicide must be evaluated i n the same dew period regime as a given bioherb i c i d e p r i o r to evaluation of combination treatments. Rates of chemical herbicides are tested at l e s s than optimum l e v e l s because of higher p h y t o t o x i c i t y observed under l a b o r a t o r y c o n d i t i o n s . Chemical h e r b i c i d e treatments to be combined with b i o l o g i c a l f



17.

BANNONETAL.

Βioherbiade Technology

Starch

Glucose Starch

311

Glucose

Liquid Medium Figure

3. E f f e c t of carbon source on e f f i c a c y and spore y i e l d of Fusarium lateritum. Spore y i e l d (Δ-Δ) was inversely r e l a t e d to v i r u l e n c e . Proprietary fermentation medium con tained starch, glucose-starch, or glucose as the primary carbon source. Spores were harvested from the fermentor and immedi a t e l y used at 10 spores/ml to inoculate p r i c k l y sida (Sida spinosa L.) i n the primary l e a f growth stage. Plants were subjected to a 20-h dew period at 25° C, removed to greenhouses for observation, and evaluated 14 days a f t e r inoculation. 6

Hours drying • i 10-h dew

^320-h dew

Figure

4. E f f e c t of propagule drying time (dew-delay) p r i o r to a 10-h or 20-h dew period on Fusarium lateritium e f f i c a c y on velvetleaf. V e l v e t l e a f plants i n cotyledon to 1-leaf growth stage were inoculated with F . lateritum spores (10 spores/ml) harvested from p e t r i plates (31 32). Dew chamber (Percival Manufacturing Co.) a i r temperature was 25° C. Dew was delayed (drying time) for indicated times. 6

r


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.Velvetleaf Mortality t%l

•120-hdew

Rain+ 20-h dew

Figure

5. E f f e c t of host (velvetleaf) p r e d i s p o s i t i o n with simulated r a i n f a l l (1.3 cm) p r i o r to inoculation at cotyledon to 1 - l e a f growth stage with Fusarium lateritium (10 spores/ml). Incubation i n a dew chamber at 25° C was delayed f o r 1 h a f t e r inoculation. Spores were produced and harvested as described previously (31 32). 6

r

100

felvetteaf Mortality M

80 604020-

L0

0.1

0.01

0

Concentration, w/v Figure

Effect of adsorbent (activated charcoal) concentration on Fusarium lateritium e f f i c a c y on v e l v e t l e a f i n o c u l a t e d at cotyledon t o 1-leaf growth stage with 10 spores/ml. Spores were produced and harvested as described previously (31 32). Incubation i n dew chamber was delayed f o r 8 h a f t e r inoculation. 6

r



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BANNON ET AL.

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Biokerbkuk Technology

herbicides may also include surfactants dependent upon the s e n s i t i v i t y of the bioherbicide to these substances. Several chemical herbicides were examined f o r e f f i c a c y on v e l v e t l e a f (Abutilon theophrasti Medic.) a f t e r placement i n a dew chamber for 20 h at 25° C. Because many chemical herbicides have a l a b e l r e s t r i c t i o n that defines a drying time p r i o r to exposure of herbicide-treated weeds to r a i n f a l l , drying time was v a r i e d p r i o r to placement i n a dew chamber. Bentazon [3-(1-methylethyl)-(IH)2,1,3-benzothiadiazin-4(3H)-one, 2,2-dioxide] activity on v e l v e t l e a f increased very slowly as drying time increased u n t i l 70% v e l v e t l e a f control was attained for those plants remaining dry f o r the e n t i r e experiment (Figure 7). Velvetleaf control by 2,4-DB [4(2,4-dichlorophenoxy) butanoic acid] reached nearly 100% a f t e r 1 h drying time (Figure 7) . In addition t o suggesting d i f f e r e n t i a l uptake of 2,4-DB and bentazon by v e l v e t l e a f , t h i s experiment c l e a r l y shows the importance of understanding response of chemical herbicides i n the experimental systems used to evaluate b i o h e r b i cides. When a c i f l u o r f e n [5-[2-chloro-4-(trifluoromethyl)phenoxy]2-nitrobenzoic acid] a c t i v i t y on v e l v e t l e a f was observed i n a dew chamber environment a f t e r exposure to increasing drying time a f t e r a p p l i c a t i o n , herbicide a c t i v i t y reached a peak a f t e r 1 h drying, slowly declined, then reached another peak at the 24 h drying period (Figure 8) . I t i s i n t e r e s t i n g to note that the l a b e l f o r a c i f l u o r f e n s p e c i f i e s a 6-h drying period a f t e r a p p l i c a t i o n f o r minimizing e f f e c t s of r a i n f a l l . Under conditions of these tests i n which chemical herbicide was applied i n 1871 L per hectare with a laboratory atomizer, treated 1-2 l e a f v e l v e t l e a f plants were placed i n a greenhouse f o r s p e c i f i e d drying times then placed i n a dew chamber f o r 20 h at 25° C; observations were made 14 days a f t e r treatment. Obviously, i n order to optimize e f f e c t s of both a chemical herbicide and bioherbicide as a combination treatment, the e f f e c t s of drying time p r i o r to an incubation p e r i o d must be thoroughly understood f o r both the b i o l o g i c a l and chemical h e r b i cide agents. E f f e c t of Spray A d j u v a n t s . The e f f e c t of a surfactant with a c i f l u o r f e n i s also shown i n Figure 8. The e f f e c t s of chemical h e r b i c i d e s i n the presence or absence of a surfactant must be determined i n experimental systems used to evaluate bioherbicides. The e f f e c t of surfactants/adjuvants on chemical herbicides are many and v a r i e d and have been reviewed recently (2£) . I t i s axiomatic that many adjuvants are t o x i c to microorganisms (37-39) thus, the i n t e r a c t i o n of spray adjuvants with bioherbicides must be evaluated on a case-by-case basis. The apparent hydrophobicity of A. cassiae spores prevents even d i s p e r s i o n i n aqueous spray systems without the use of a surfac tant. Although non-ionic surfactants such as Sterox NJ {nonoxynol (9 to 10 POE) [α-(p-nonylphenyl)-ω-hydroxypoly (oxyethylene)]} have been used to disperse A. cassiae spores (JJL), i t was found that e m u l s i f i e d o i l s are superior to non-ionic surfactants f o r t h i s purpose (1£) (Figure 9). This may be a t t r i b u t e d to the fact that binding of A. cassiae spores to the host i s superior when emulsi f i e d o i l s 1% (v/v) are included i n the spray solution. When drying time following host i n o c u l a t i o n with A. cassiae spores i s varied p r i o r to 0.6 cm simulated r a i n f a l l , e f f i c a c y of t h i s bioherbicide on s i c k l e p o d i s increased i n the presence of e m u l s i f i e d o i l s compared to non-ionic surfactants (Figure 10). The adhesive e f f e c t of o i l s , surfactants, and other spray adjuvants i s known (4JJ . r



314


04—ι 0 1

1 3

1

1 6

1

1 12

1

1 24

1

1 Infinity

Drying Time, Hours 2,4-oa 0.3 kg/ha

bentazon, 0.6 l ^ a

Figure 7. E f f e c t of drying time p r i o r to dew chamber incuba t i o n f o r 20 h at 25° C on 2,4-DB or bentazon e f f i c a c y on velvetleaf. Observations were made 14 days a f t e r treatment, and herbicides were applied at rates indicated i n 1871 L/ha. Herbicide solutions contained X-77 surfactant, 0.25% (v/v).

Velvetleaf Mortality Cfl 80-

0 -I 0

1 1

1 3

1

1 6

1

1 12

1

1 24

1

1 Infinity

Drying Time, Hours acifluorfen

•·«•• acifluorfen + Ag 98

8. E f f e c t of drying time p r i o r to dew chamber incuba t i o n (20 h) on a c i f l u o r f e n e f f i c a c y on v e l v e t l e a f . Triton AG98 i s a non-ionic surfactant, and i t was added to the diluent at a rate of 0.25% (v/v) where i n d i c a t e d . Herbicides were applied to 1-2 l e a f velvetleaf plants i n 1871 L/ha and observa tions were recorded 14 days after treatment.

Figure



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Figure 9. E f f e c t of non-ionic surfactants 0.25% (v/v) (X-77, Sterox NJ AG-98) or emulsified o i l s 1% (v/v) (Agri-Dex SoyDex) on e f f i c a c y of Alternaria cassiae (1.1 kg/ha) under f i e l d conditions. Sicklepod plants i n cotyledon t o 1-leaf growth stage were inoculated with A. cassiae spores i n 337 L/ha. No dew formation occurred f o r 24 h a f t e r i n o c u l a t i o n . Overhead i r r i g a t i o n (0.6-1.3 cm) was applied to plots 24 h a f t e r inocu lation. Observations were recorded 4 weeks a f t e r treatment. Spores were produced by a p r o p r i e t a r y m o d i f i c a t i o n of the method of Walker (!£) . f

f

Drying Time, Hours • i Sterox NJ

E2ax-77

W AgriOex

S 3 Sun Oil IllNl

Figure 1 0 . Effect of drying time p r i o r to simulated r a i n f a l l χ spray adjuvant i n t e r a c t i o n on e f f i c a c y of Alternaria cassiae (1.1 kg/ha). Spores were produced by a proprietary modifica t i o n of the method of Walker (JJL) · Sicklepod p l a n t s i n cotyledon to 1-leaf growth stage were i n o c u l a t e d with A. cassiae spores i n 1871 L/ha containing 0.25% (v/v) Sterox NJ or X-77; 1% (v/v) Agri-Dex or Sun O i l (UN). After indicated dry ing times inoculated plants were subjected to 0.6 cm simulated r a i n f a l l and placed i n a dew chamber f o r 8 h at 25° C. Plants were removed to a greenhouse and observations were recorded 14 days a f t e r inoculation. Hoagland; Microbes and Microbial Products as Herbicides ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

316



Recently, Quimby et al. (42.) have shown that an i n v e r t emulsion g r e a t l y improves e f f i c a c y of a bioherbicide, A. cassiae, i n the absence of free moisture. Further research i s i n progress to evaluate e f f e c t s of i n v e r t emulsions and t h e i r formulation compo nents on bioherbicide e f f i c a c y , e s p e c i a l l y i n the absence of free moisture. E f f e c t s nf Tptnpprat-nrp Walker and R i l e y (15.) characterized the e f f i c a c y of A. cassiae on sicklepod under d i f f e r e n t temperature regimes. Optimum e f f i c a c y was observed at 25° C, and e f f i c a c y d e c l i n e d s i g n i f i c a n t l y at 35° C. It would be advantageous f o r a bioherbicide to control i t s respective host(s) over a wide tempera ture range. Laboratory t e s t s (unpublished data) have shown that A. cassiae spores germinate at 35° C on the host t i s s u e but do not i n f e c t or k i l l the host. A. cassiae i s a necrotrophic b i o h e r b i c i d e , which, conceptually, means that a t o x i n i s produced i n advance of the colonizing bioherbicide (42.) · The bioherbicide then l i v e s on dead plant t i s s u e as a saprophyte. The AK-toxin produc t i o n c h a r a c t e r i s t i c of Alternaria alternata i s i n a c t i v a t e d by heat (A4.) . Perhaps a s i m i l a r phenomenon i s occurring i n A. cassiae although s p e c i f i c toxins responsible f o r observed sicklepod phytot o x i c i t y have not been i d e n t i f i e d . I t should be noted that Hradii et a l . (45.) recently i d e n t i f i e d four phytotoxins from A. cassiae, and a l l of these compounds exhibited a low l e v e l of phytotoxicity to sicklepod. Further research i s required to understand metabolic c o n t r o l of processes r e l a t i n g to v i r u l e n c e and pathogenicity of bioherbicides. Source-Sink Relationships. C e l l u l a r sugar l e v e l s often influence pathogenicity of c e r t a i n diseases. Glucose i n h i b i t i o n of carbohydrase production by Alternaria solani ( E l l . and G. Martin, L. R. Jones and Groust) was reported by Sands et a l . ( 4 £ ) · Carhohydrases have been reported to be involved i n mechanisms of pathogenicity (47-50). The fact that cytokinins can simulate the accumulation of nutrients at the s i t e of a p p l i c a t i o n i s well-known by plant scien t i s t s ( 5 1 ) . In order to simulate a sink e f f e c t , l e a f l e t s on one side of a mature sicklepod l e a f were treated with a 30 μΜ s o l u t i o n of k i n e t i n ; whereas, l e a f l e t s immediately opposite were t r e a t e d with the diluent only used to d i s s o l v e the k i n e t i n . The l e a f was then treated with a 1.1 kg/ha (10 spores/ml) rate of A. cassiae and incubated i n a dew chamber for 8 h at 25° C. It was observed that the k i n e t i n - t r e a t e d l e a f l e t s f a i l e d to develop severe necro s i s , whereas, the untreated opposite l e a f l e t s developed n e c r o t i c symptoms and senesced. A second experiment was i n i t i a t e d to examine the e f f e c t s of a photosynthesis i n h i b i t o r to decrease s i c k l e p o d l e a f sugar l e v e l s followed by an a p p l i c a t i o n of A. cassiae spores. When the photosynthesis-inhibitor, l i n u r o n , was a p p l i e d to sicklepod and followed 3 to 5 days l a t e r with a 1.1 kg/ha a p p l i c a t i o n of A. cassiae, e f f e c t i v e control of 3 to 5 l e a f sicklepod was obtained (Table I ) . Normally, A. cassiae i s e f f e c t i v e only on cotyledon to 1-leaf sicklepod plants. Although l e a f sugar l e v e l s were not analyzed, these data suggest that further research should be i n i t i a t e d to explore interactions between i n t e r mediary metabolites and enzyme systems which i n f l u e n c e patho g e n i c i t y of bioherbicides. s


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Table I. E f f e c t of Alternaria cassiae (1.1 kg/ha) on Sicklepod Control i n Simultaneous or Sequential Combination With Linuron (0.1 kg/ha)

Rate, kg/ha

Treatment


1

Sicklepod Mortality, %

A. cassiae

1.1

10

linuron linuron + A. cassiae linuron + A. cassiae

0.13 0.13+1.1 0.13+1.1

20 40 85

2

3

1

2

3

Sicklepod plants inoculated with A. cassiae spores suspended i n water containing emulsified crop o i l 1%, v/v (MYD 751, Mycogen Corporation proprietary) . Diluent containing spores was applied at 1871 L/ha to sicklepod plants i n the 3 to 5-leaf growth stage. Dew period immediately following inoculation was 6-h at 25° C. A. cassiae spores tank-mixed with linuron. A. cassiae spores a p p l i e d as i n footnote 1 but 3 days a f t e r linuron application.

Summary Successful discovery and development of novel b i o h e r b i cides w i l l continue to be dependent upon an integrated research strategy i n v o l v i n g both public and private sectors. Development of products which perform consistently w i l l be based on an understanding of the bioherbicide and i t s i n t e r a c t i o n with the host and i t s environment. Research to-date suggests that free moisture i s the most important environmental v a r i a b l e i n f l u e n c i n g c o n s i s t e n t bioherbicide efficacy. However, other f a c t o r s which a f f e c t propagule production, s t a b i l i z a t i o n , pathogenicity, and virulence must be examined to successfully develop bioherbicides. The i n f l u ence of proper market focus should not be e x c l u d e d i n research/commercialization strategies. Moisture i s a key component of the plant disease t r i a n g l e . As such, i t influences propagule s t a b i l i t y through physiology of rehydration and dehydration. Proper moisture removal a f f e c t s long term propagule storage. Interaction of free moisture (dew) with the bioherbicide propagule determines herbicide e f f i c a c y on the host. Although other f a c t o r s such as spray adjuvants, p r e d i s p o s i t i o n , p h y s i o l o g i c a l state of the host, and chemical herbicide i n t e r a c t i o n s also influence bioherbicide e f f i c a c y , predominant e f f e c t s on host-pathogen i n t e r a c t i o n s are e x h i b i t e d by moisture. A proper understanding of bioherbicide physiology and biochemistry as i n f l u enced by moisture and other environmental variables w i l l r e s u l t i n more consistent, e f f i c a c i o u s bioherbicides.

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