broomrapes ( orobanche spp . ) are fully parasitic flowering plants that lack chlorophyll ; hence they penetrate roots of susceptible hosts , removing water , minerals and sugars .
( egyptian broomrape ) attacks dicotyledonous crops cultivated around the mediterranean , causing massive yield losses .
broomrape attached to the host by means of tubercle , a swollen organ which may be simple or composite . with the exception of the case of transgenic target - site , herbicide - resistant host plants , meant to be a temporary measure until other effective control means are found .
orobanche generally maintains a close relationship with the host and so it is unreasonable to attack it using herbicides because the latter may adversely affect the nontarget host . despite research on orobanche spp .
for over three decades , yield losses still abound because there is no sustainable method for controlling the parasite .  
 fusarium compactum ( wollenw . )
the inundative biocontrol approach , with repeated applications of the biocontrol agent , generates a state of equilibrium with a very low level of weed density as a result of the artificial inoculation of the biocontrol agent but the fungi are not sufficiently virulent for field release , regardless of the amount used .
a series of experiments were conducted using f.   compactum , a biological control agent that infects orobanche without affecting the roots of tomato .
pectins are complex polysaccharides and are one of the major components of the plant cell wall of dicotyledonous plants , where they control the ionic status , cell expansion , and separation .
some cellulase producing fungi includes acremonium sp , aspergillus spp and fusarium sp , , trichoderma spp .
[ 6 , 7 ] , zymomonas , and mutant penicillium . in this paper ,
a hydrolytic enzyme plays an important role in the pathogenicity of plants by facilitating fungal penetration through the host cell wall [ 10 , 11 ] .
experiments using mycoherbicidal organisms plus pectinase ( ec 3.2.1.15 )   or cellulase ( ec 3.2.1.4 )   indicate that enzyme enhances the weed control of pathogenic fungi . here
, pectinolytic and cellulolytic enzymes have been used to enhance the virulence of f.   compactum on tomato plants infested with broomrape .
a semiaxenic polyethylene bag system was used that allowed easy visual observation of the fungal infection of the tubercles .
this study reports that the addition of pectinase and cellulase alone or in mixtures enhanced the virulence of f. compactum on broomrape .

 f. compactum was cultured on potato glucose agar ( pda , pronadisa ) in petri dishes incubated at 25c .
subcultures were grown in 100  ml potato glucose broth ( pdb , pronadisa ) in 250  ml erlenmeyer flasks .  
the cultures were left on a rotary shaker ( brunswick scientific ) at 150  rpm for 48  h. f.   compactum mycelia were harvested on miracloth ( calbiochem , la jolla , ca ) , rinsed with distilled water to remove remaining spores and excess medium , and harvested by vacuum filtration .
the washed hyphae were chopped at 6,000  rpm for 2  min with a homogenizer ( ika t18 basic ultra - turrax usa ) , resuspended in sterile water , and the propagule concentrations of chopped mycelia were estimated after serial dilution and plating .
surface sterilizing seeds ensure that the fungal infection on the seeds is from deliberate infection .
thus , about 13  mg seeds in small bags formed of miracloth were wetted and surface sterilized in 80% ethanol for 1  min and in a mixture of 1% sodium hypochlorite in 0.01% aqueous tween 20 for 10  min .  
tomato transplant plugs at the two to three leaf stages in speedling insert trays were purchased from hishtil inc , ashkelon , israel .  
the pathogenicity of f.   compactum was tested in the semiaxenic polyethylene bag system ; briefly about 13  mg of dry surface - disinfected seeds ( up to 1,500 ) were sprinkled on wet whatman gf / a glass - fiber sheets ( whatman int .  
the broomrape seeds were conditioned for a 7  d period on the wet glass - fiber sheets .
a tomato seedling with three or four expanded leaves and washed roots was fixed inside each polyethylene bag containing conditioned broomrape seeds .
the plant roots in each bag were moistened by capillary action with forty  ml of modified hoagland 's solution in the base of each bag . modified hoagland 's solution
fourteen - hour photoperiods were provided by a photosynthetically active light intensity of 65  e / m / s ( li - cor , inc . , photometer , model li-188b ) produced by six 40  w cool white fluorescent tubes suspended 35 cm above the benches . two  ml of 5-g  ml gr-24 ( synthetic germination stimulant ) were added to each bag with a pipette to augment the tomato root exudates .
the broomrape seeds germinated , attached to tomato roots , and formed small tubercles during the following 2 weeks .         
allocation of treatment to orobanche - infested tomato plants were in such a way that the tubercle numbers and sizes were almost the same .
the virulence of the fungus was determined with and without various concentrations of either pectinase ( ex fungal origin , 1.1  u  mg , sigma ) and/or cellulase ( cellulysin , ex trichoderma viride , 10  u  mg calbiochem - behring corp . , la jolla , ca 92037 ) .
the effect of cellulase concentration ( 10 to 20  u  ml ) on tubercle death was similarly determined at a constant inoculum level . thereafter , the virulence of the fungus with the two enzymes was determined in combination at varying ratios .  
control plants were mock - inoculated with either sterile distilled water containing 0.01% tween 20 or 4 to 20  u  ml of single or combined enzyme preparations but without fungal mycelia .
tubercles on the tomato plants infested with broomrape were counted and the diameters were measured with a ruler , with the assumption that the tubercles are perfectly spherical . the treatments consisted of f. compactum or f. compactum plus cellulase ( 4 to 20  u  ml ) .
one  ml of aqueous fungal cellulase samples ( 10  units  mg ) , freshly made for each experiment as a solution containing 10  u  ml , and was checked for pectinase activity .
pectinase activity was measured in a reaction mixture consisting of 533  l of 1% polygalacturonic acid ( pectin ) , 400  l of 50  mm sodium acetate buffer at ph 5.0 and 67  l of the cellulase .
the mixture was incubated at 37c for 10  min as outlined by tonukari et al . .
a 100-l aliquot of the reaction mixture was mixed with 1.5  ml of 1% 4-hydroxybenzhydrazide ( fluka , fluorescence grade ) in 0.5  m  naoh .
the mixture was heated at 100c for 10  min , and cooled on ice water .
absorbance was measured at 410  nm against the zero time blank as outlined by lever , using a spectrophotometer with a versamax tunable microplate reader .
pectinase activity of the cellulase was calculated from a standard curve prepared with a d - galacturonic acid ( sigma ) .
one unit of enzyme forms 1  mol of galacturonic acid from polygalacturonic acid in 1  min under the conditions of the assay .
broomrape tubercle deaths were recorded at 24  h intervals after fungal inoculation , for 8 to 11  d.   tubercles were visually scored as healthy ( translucent , dense , and intact ) , infected ( diseased ) , or dead ( black and soft ) .
the levels of infection caused by f.   compactum plus pectinase in both experiments were always better than those achieved by the fungus without pectinase ( figures 1(a ) and 1(b ) ) .
the numbers of broomrape tubercles infected continuously increased over time throughout the period of observation .
this was not only due to inoculum buildup in the tomato root system but also to the enzyme action .
this study wanted to ascertain whether the added pectinase would have an effect on suboptimal f.   compactum inoculum . therefore
, subthreshold levels of inoculum ( 10  propagules  ml ) were investigated in the presence of pectinase .
f.   compactum alone killed 30% of broomrape tubercles at the suboptimal inoculum levels ( figure 1(a ) ) . at the lowest inoculation density ,
f.   compactum ( 3.84  10  propagules  ml ) combined with 11  u  ml pectinase killed more broomrape tubercles than the f.   compactum alone ( figures 1(a ) and 1(b ) ) .
the killing of broomrape tubercles indicated that pectinase enhanced the virulence of f.   compactum ( figure 2 ) .
since both enzymes separately enhanced the severity of tubercle infection , it was ascertained whether the pathogenicity of f.   compactum ( 1.4  10  propagules  ml ) on broomrape tubercles could be synergistic through the joint action of the enzymes ( 10  u  ml of each enzyme ) ( figure 3 ) .
the broomrape tubercles on the tomato roots were large and healthy in the absence of fungal treatment but , at times , a few naturally brown tubercles could be seen ( figure 3(a ) ) .
f.   compactum alone infected all the inoculated tubercles but did not kill any significant number ( figure 3(b ) ) .
infested tomato roots that were inoculated with mycelia plus pectinase ( 20  u  ml ) ( figure 3(c ) ) had over 50% tubercles dead one week after treatment .
those inoculated with mycelia plus cellulase ( 20  u  ml ) ( figure 3(d ) ) had above 60% mortality .
a mixture of both enzymes with f.   compactum increased fungal infection of broomrape by f.   compactum ( figure 3(e ) ) .
the response of the infected tubercles varied from 100% kill to mild infection , depending on tubercle size .  
mycelia plus cellulase ( 20  u  ml ) mix provoked about the same level of infection as mycelia in solution with cellulase and pectinase ( 10  u  ml of each enzyme ) ( figure 3(f ) ) .
it is logical to think collective mixing of pectinase and cellulase to f. compactum will effectively enhance the fungal biocontrol potential : thus , it was tested . at various enzyme compositions ,
the f. compactum treatment ( 1.05  10  propagules  ml ) that caused only a hypersensitive reaction ( 9% death ) on the tubercles caused about 35 to 85% tubercle death when the tubercles were treated with mixtures that contained mycelia , pectinase , and cellulase in various ratios ( figure 4 ) .
higher tubercle infection by f. compactum was observed with a high ratio of cellulase to pectinase .  
beginning from day 4 after f. compactum inoculation on broomrape infested tomato plants ; statistically significant differences among treatments were observed ( figure 4 ) .
broomrape infested roots coinoculated with chopped mycelia plus pectinase and cellulase had substantial tubercle infection and subsequent large numbers of dead tubercles ( 3585% ) ( figure 4 ) .
this system ensures easy examination of seedling attachment to the tomato roots and easy observation of tubercle infection in the course of the experiment .
the activity catalyzed by an enzyme is a measure of the amount of enzyme present .
values pooled from three sets of experiment showed that in a 1  mg ( 10  u  mg ) cellulase preparation , 0.055  mg pectinase ( 1.1  u  mg ) is present .
this means the 10  u cellulase has a 0.06  u of detectable pectinase .
the basis for this study stems from previous studies   which examines single enzyme - assisted effect on fungi pathogenicity .
the orobanche were at three main phases of their life cycle ( seed , germination , and parasitic phases ) at fungal inoculation .
tubercles are usually present at the germination phase and later become parasitic . in this study ,
in apparently healthy tubercles , it may be possible to find diseased tubercles ( as depicted in figure 2 ) ; such tubercles deaths were induced by accidental infestation . in total , these results demonstrate that exogenous pectinase as well as cellulase can contribute to pathogenicity .
the mycelia plus enzyme mix killed some broomrape tubercles , thus they could not form new seeds to replenish the orobanche seed bank .
the rationale of approach is also applicable to soil grown tubercles where the presence of enzyme may be exogenous .
this is well taken care of in nature because the tubercles are borne on the roots and roots are mostly below the soil surface .
the temperature in the growth chamber was kept at 25c to induce infection by f. compactum .   as demonstrated in this study ,
descriptions of actions of cell wall degrading enzymes are given by wanjiru et al . .
f. compactum can infect the tubercle by degrading the cell wall components and invading the tissues and cells .
pectinolytic and cellulolytic activities of the enzymes ( in this case of fungal origin ) result in a loss of structural integrity in the tubercle and characteristic damage .
the enzyme substrate range and mechanism of action could partly explain the interaction between f. compactum and the enzymes on broomrape tubercles .
the pathogenicity of f. compactum plus the enzyme mix observed could rarely be observed without pectinase and cellulase at the inoculum level used .
besides , neither pectinase nor cellulase was ordinarily able to injure and kill broomrape at the concentration used ( max . 20  u  ml ) , demonstrating that the broomrape tubercle infection and death observed are due to the addition of a mycoherbicidal organism .
the role of pectinase and cellulase in the degradation of cell wall material as observed in this study corresponds with the reports of many authors [ 10 , 11 , 18 , 19 ] . in the previous paper ,
the results reported by sasaki and nagayama   showed that the fungi pathogenicity was not always proportional to the enzyme activity .
it is known that the level of -glucosidase in an enzyme preparation may affect the result of cellulase assays , for example , for the estimation of activities of extracellular cellulase enzymes produced by trichoderma . as pointed out by kumpoun and motomura
, the pectinase used in their study is mainly pectinase , but some glycosidases were also present .
the presence of -glucosidase or other enzymes , such as cellobiose phosphorylase , which are required for cellobiose metabolism and to enhance cellulose hydrolysis but which are not , strictly speaking , cellulases or rhamnosidase and -glucosidase activities in pectinase   could further complicates research findings .
care must be taken in interpreting the results of pathogenicity tests as the enzyme preparation may contain a battery of enzymes .
this result may suggest the need to study the presence of other enzymes in an enzyme of interest even if the enzyme of interest is from a commercial source .     
contrary to some of the findings associated with enhanced f.   compactum infection on broomrape , f. oxysporum pathogenicity on broomrape was not enhanced by pectinase and/or cellulase .
thus , although f. oxysporum and f.   compactum belong to the same genus , they may have different mycoherbicidal mechanisms .
mycelia of f.   compactum did not penetrate nor show apparent damage to the tomato roots   as also observed in this study . in conclusion , pectinolytic and cellulolytic activities are widely exhibited by bacteria and fungi .
the enzymatic activities can predispose broomrape tubercles to infection by fungi , in this case , f.   compactum .
the latter fact adds to the commercial value of f.   compactum as a potential mycoherbicide when sufficiently virulent .
 the use of enzyme could facilitate pathogen penetration into plant host . here 
 the combination of cellulase and pectinase was ascertained on the pathogenicity of f. compactum ( 1.4  106  propagules ml1 ) on broomrape tubercles . 
 f.   compactum alone infected all the inoculated tubercles but did not kill any significant number . 
 infested tomato roots that were inoculated with mycelia plus pectinase ( 20  u  ml1 ) had over 50% tubercles dead one week after treatment . those inoculated with mycelia plus cellulase ( 20  u  ml1 ) had above 60% mortality . 
 mixtures of mycelial plus the two enzymes ( 10  u  ml1 of each enzyme ) showed synergy . 
 the activity catalyzed by an enzyme is a measure of the amount of enzyme present . 
 it was shown that , in a 1  mg ( 10  u  mg1 ) cellulase used , 0.055  mg pectinase ( 1.1  u  mg1 ) is present . 
 this explains why mycelial plus cellulase mix contends with mycelial plus the two enzymes . 
1. Introduction
2. Materials and Methods
3. Results and Discussion