Evaluation of Fusarium as potential biological control against Orobanche on Faba bean in Tunisia
M. Zouaoui Boutiti,1 T. Souissi1 and M. Kharrat2
Summary A total of 149 fungal strains identified as Fusarium were isolated from infected Orobanche crenata Forsk. and Orobanche foetida Poir. plants. Their pathogenicity and virulence were assessed in Petri dish assays using lentils as the medium. Ten isolates were found to reduce the number of tubercles at- tached to the host plant. Among them, two isolates that caused necroses on tubercles of Orobanche in the Petri dish assays were identified as Fusarium F6 and F10. They reduced the number of tubercles of O. crenata by 97% and 98%, respectively. Inoculums of F6 and F10 were produced on barley grains and were tested in sterilized and non-sterilized soil in separate pot experiments, using O. cre- nata and O. foetida as parasitic plants. Both isolates reduced the number of O. crenata and O. foetida by 68% and 88%, respectively, and their dry matter by 82% to 88%. A similar experiment conducted using formulated inoculums of the two isolates showed that the formulation improved the efficiency of the fungi, and reductions in the number and dry matter of tubercles to 100% were observed. These results suggest that Fusarium isolates have the potential to be used as biological control agents against O. crenata and O. foetida on faba bean in Tunisia.
Keywords: pathogens, pathogenicity, virulence, parasitic plants, broomrapes.
Introduction the numerous tiny seeds that retain their viability in the soil for 6 to 20 years. Germination of Orobanche seed Broomrapes, Orobanche spp., of the family of Oro- requires a stimulant excreted by the host plant and pro- banchaceae are troublesome root parasitic weeds duces germ tubes that attach to the host plant (Raynal that cause severe damage to vegetables, legumes and et al., 1989). The germ tube develops a haustorium and sunflower (Parker and Riches, 1992). Approximately, forms a tubercle. The haustorium represents the phy- 16 million hectares of arable land in the Mediterrane- sical and morphological contact between the parasite an region as well as in west Asia are currently endan- and the host. It supplies the parasite with water, mineral gered by Orobanche infestation (Sauerborn, 1994). In nutrients and organic materials from its host (Kroschel, Tunisia, Orobanche crenata Forsk. distributed in the 2001). north-east and O. foetida Poir. in the north-west, are So far, no efficient control measures for Orobanche the main species that cause losses in leguminous crops, spp. have become available to farmers (Müller-Stöver, especially on faba bean (Kharrat and Halila, 1994). 2001). Single methods such as delayed sowing and Losses in faba bean fields can reach 80% (Kharrat, use of resistant varieties have shown unsatisfactory re- 2002). Difficulties in controllingOrobanche are due to sults. The use of chemical products such as glyphosate requires care to avoid phytotoxicity. Thus, Orobanche represents a difficult target for selective chemical con- 1 National Agronomic Institute of Tunisia, 43 Avenue Charles Nicolle, trol. Control of Orobanche may be possible by integra- 1082 Tunis-Mahragène, Tunisia
238 Evaluation of Fusarium as potential biological control against Orobanche on Faba bean in Tunisia
Research has been conducted in several countries in Bioassays cluding Algeria, Egypt, Germany, Maroc and Chili The isolated fungi were evaluated to assess their (Klein et al., 1999; Zermane et al., 1999; Müller-Stöver, phytotoxic ability on the growth of the underground 2001; Boari and Vurro, 2004). Fusarium oxysporum f. stages of Orobanche. In these Petri dishes bioassays, sp. orthoceras (Appel and Wollenw.) Bilai obtained seeds of O. crenata were used as the parasitic weed from diseased O. cumana tested in soil with sunflower and those of lentil were used as the host plant. The me- as a host plant was able to reduce the number of attached thods followed those of Kroschel (2001). Plastic Petri and emerged broomrape seedlings by about 90% (Bedi dishes were filled with washed sterile sand, watered and Donchev, 1991). F. oxysporum f. sp. orthoceras on and covered with filter paper.Orobanche crenata seeds O. cumana was also tested by Thomas et al. (1998). were sterilized with sodium hypochlorite, rinsed with Recently, F. arthroporioides and F. oxysporum isolated distilled water and sprinkled on the filter paper at the in Israel from O. aegyptiaca were shown to be effective densities of 25 seeds per square metre. The Petri dishes in reducing broomrape growth (Amsellem et al., 2001). were covered with black plastic and incubated in the The pathogenicity of two isolates, Ulocladium botrytis dark for conditioning at 22°C for 10 days. To enhance Preuss and F. oxysporum Schlecht. f. sp. Orthoceras, pre-conditioning in the Orobanche seed, 100 ppm of were tested by Müller-Stöver (2001). The two fungi cause gibberellic acid was added. Pre-germinated lentil seed necroses on both O. cumana and O. crenata. Currently, lings were inserted into sand in the Petri dishes through the development of an appropriate formulation whi- holes made in the surface of the filter paper. ch allows successful application of fungal propagules To test the pathogenicity of fungal isolates, fresh col will determine the success of Fusarium in agriculture onies of the isolated fungi growing on SNA medium applications. The encapsulation of fungal propagules were used. For each Petri dish, the black plastic was in a solid matrix ‘Pesta’ was used by (Müller-Stöver, removed and filter paper containing O. crenata seeds 2001). A 70% reduction of Orobanche emergence was and lentil seedlings were sprayed with 10 ml of the obtained when wheat flour kaolin granules containing spore suspension at 106 spores per millilitre. The Pe- chlamydospore rich biomass was applied. tri dishes were incubated in the green house at 25°C Considering the importance of O. crenata and and 16 h/18 h photoperiod for 5 weeks. Two replicates O. foetida in Tunisia and the lack of research on fungi were used per treatment. The number of germinated, associated to Orobanche spp., the main objective of this attached seeds and the number of tubercles formation study was to screen and evaluate the potential of fungi were recorded. isolated from Orobanche with potential as biological control agents against the parasitic weed, in laboratory and green house experiments. Test of specificity The isolates which reduced the number of tuber- Materials and methods cles of Orobanche in Petri-dish assays were selected and were tested for their host specificity. A range of Field surveys plants was used that included tomato (Lycopersicon Field surveys were carried out from April 2004 to esculentum Mill), carrot (Daucus carota L), Faba May 2005 in northern Tunisia, especially in the region bean (Vicia faba L. and Vicia faba L. minor), pea (Pi- of Nabeul. Underground stages of Orobanche with sum sativum L.), chickpea (Cicer arietinum L.), lentil symptoms of fungal infections such as browning and (Lens culinaris L), wheat (Triticum aestivum L.) and rotting were collected. The plants were conserved in barley (Hodeum vulgare L.) These plants were gro- laboratory until use. wn in pots in a greenhouse. Ten days after emergence roots were washed with distilled water and immersed 6 Isolation in the inoculums at the concentration of 10 spores per millilitre, for 5 to 10 min. Then plants were transplan- Isolations were made from pieces of tubercles and ted in pots and observed weekly for 1 month for the stems with fungal symptoms. Diseased tissues were ex- development of symptoms. Four plants of the same cised, washed with distilled water, sterilized in 1% so- test plant species were used per pot with four pots per dium hypochlorite with Tween 20 for 5 min and rinsed tested plant. four to five times with sterile distilled water. After dry- ing on filter paper, pieces were placed in Petri dishes on potato dextrose agar (PDA™) medium supplemented Pot assays with 100 ppm of streptomycin. The Petri dishes were Selected fungal isolates that were able to reduce incubated in the dark at 22°C until fungal development Orobanche seed germination in the Petri dish assays occurred. Repeated sub-culturing was done to obtain were tested in sterilized and non-sterilized soils in pots pure cultures. Isolates were conserved on special nutri- in a greenhouse. Both O. crenata and O. foetida were ent poor agar (SNA) at 5°C for short term storage and used as parasitic weeds and V. faba as the host plant in liquid nitrogen for long-term storage. during this experiment.
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Preparation of inoculum: Barely grains were used as mixture was kneaded with gloved hands and passed a solid inoculum. Ten ml of sterile water were added to through a small, hand-operated pasta maker. Obtai- fresh colonies of the fungi growing on SNA medium. ned granules were incorporated in sterilized and non- The resulting suspension, containing spores and myce- sterilized soils. The granules were added and mixed lium, was used to inoculate the organic substrate which into the soil together with the seeds. Each pot was filled was then incubated for 14 days at 25°C. with either 11 mg of O. crenata or 9 mg of O. foetida Inoculation and pot trials: Two pot experiments, us- and 1 g of granules per kg of soil. Three seeds of V. faba ing sterilized and non sterilized soils, were conducted minor were planted per pot and thinned afterwards to under greenhouse conditions to study the ability of obtain one host plant per pot. In total, five pot experi- fungi to control O. crenata and O. foetida in V. faba ments were carried out. plants. In both experiments, plastic pots with 750 g capacity were filled with a mixture of soil and sand Statistical analysis at a ratio of 2:1. Orobanche seeds were sprinkled All pots experiments were conducted in totally ran- onto the soil surface. The inoculum was added and domized design. Statistical analyses were performed mixed into the soil together with the seeds. Each pot using analysis of variance (ANOVA) with alpha 0.05 in was provided with either 11 mg of O. crenata seed or GEN-STAT software. 9 mg of O. foetida seed, or 7.5 g of the solid inoculum. Three seeds of V. Faba minor were planted per pot and thinned afterwards to obtain one host plant per Results pot. The pots were fertilized and watered each week. Five controls were prepared: the host plant alone Field survey and isolation of fungi (H), the host plant with O. crenata or host plant with One hundred and forty nine isolates were obtained O. foetida, the host plant with non-inoculated sub- from infected O. crenata plants collected during field strate (H + NIS) and host plant with inoculated sub- surveys. All isolates were found to belong to the genus strate (H + NIS). The experiments continued for Fusarium after microscope examination. 5 months and terminated when the host plant in the control was dead. The parameters used to assess the Bioassays effect of fungi on the control of O. crenata and O. foetida were faba bean height (cm) and dry matter Ten isolates of the genus Fusarium reduced the ger- weight (g) and Orobanche number and dry matter mination of O. crenata 27% to 93% and eight reduced weight (g). attachment (22% to 79%) to the host plant by germinated Orobanche (Table 1). The two exceptions, F4 and F7, did not differ statistically between the treatments and Formulation in pot experiments the non-inoculated control. The number of tubercles Wheat flour kaolin granules were prepared after the developed by O. crenata was reduced by 78% to 98% methods of Connik et al. (1991, 1996). Durum wheat (Table 1). Symptoms of necroses were observed on flour 32 g, kaolin 6 g and sucrose 2 g, were blended Orobanche inoculated tubercles. and poured into a dish. Inoculums were added as chla- Isolates F6 and F10 were the most efficient in re- mydospore in 23 ml PDB (potato dextrose broth). The ducing the percentage of tubercles of O. crenata by
Table 1. Effect of a conidial suspension of Fusarium on the development of the underground stages of Orobanche crenata in Petri dishes including; the number of O. crenata seeds that germinated, the number of germinating seeds that attached to the host and the number of tubercles formed. Isolates Number Percent reduction Number Percent reduction Number of Percent reduction germinated in germination attached in attachments tubercles in tubercles Control 36.5 – 27 – 47 – F1 7.5 79 5.5 79 7.5 84 F2 3.5 90 18 33 10 78 F3 5 86 10.5 61 2 95 F4 3 92 29 – 7.5 84 F5 5 86 9 67 5 89 F6 26.5 27 13.5 50 1 98 F7 3.5 90 24.5 9 4.5 90 F8 4 89 13 52 4 91 F9 2.5 93 13.5 50 7 85 F10 23 37 21.5 22 1.5 97 LSD(5%) 5.74 – 4.03 – 3.5 –
240 Evaluation of Fusarium as potential biological control against Orobanche on Faba bean in Tunisia
97% and 98% respectively. These isolates were used Faba bean was also increased by 35% to 45% and 79% in sterilized and non sterilized soil on O. crenata and to 82%, respectively, compared to the infested control O. foetida using faba bean as a host plant. (H + IS). Non-sterilized soils: Isolates F6 and F10 incorporated Test of specificity with barley grains as inoculum substrate into the soil reduced the number of both O. crenata and O. foetida Isolates F6 and F10 were selected for use in speci- by more than 90% compared to the infested Orobanche ficity test because in preliminary tests no symptoms control (Table 3). So, the dry matter of both O. crenata and no death were observed on test plant species. and O. foetida was increased by 100%, which did not statistically differ from both control of O. crenata and O. foetida (H + O + NIS) (Table 3). Pot assays There were no significant effects on the height or dry matter of faba bean compared to either Orobanche Sterilized soil: F6 and F10 tested in sterilized soil on species (Table 3). High significantly, no tubercles were O. crenata and O. foetida reduced the number germi- produced by either species. nated and dry matter weights of both parasitic plants. In O. crenata the number of tubercles was reduced by 70% to 87% compared to the infested the controls (H + Formulation experiments in pots O + NIS), and the dry matter of tubercles was reduced There were no tubercles produced and therefore no by 88% (Table 2). Inoculation with F6 and F10 iso- tubercle dry matter for O. crenata and O. foetida when lates resulted in 36% to 38% increase in height of faba wheat flour kaolin granules containing chlamydospore bean compared to the Orobanche infested control (H + rich biomass was applied in sterilized or non sterilized IS). The dry weight of the host plant was also signifi- soils (Figure 1). cantly increased by 120% to 129% compared to the Orobanche-infected control (Table 2). Discussion Isolates F6 and F10 reduced the number of O. foetida by 68% to 77% whereas the dry matter was reduced The use of Petri dishes allowed observation of the under- by 81% to 84% compared to the infested control (H + ground stages of Orobanche (germination, attachments O + NIS) (Table 2). The height and the dry matter of and tubercles) which would not have been possible
Table 2. Effect of isolates F6 and F10 on the number of tubercles and tubercle dry weight of Orobanche crenata and O. foetida and on the height and dry weight of faba bean, in sterilized soil. Treatments Plant height Percent Plant dry Percent No. of Percent Tubercles dry Percent (cm) increase matter (g) increase tubercle reduction matter (g) reduction Vica faba minor, faba bean Orobanche crenata Control (H) 64.8 – 5.39 – – – – – Control (H + IS) 65.4 – 5.65 – – – – – Control (H + F6) 89.4 37 9.41 66 – – – – Control (H + F10) 89 36 8.6 52 – – – – Control (H + O) 51.4 – 2.66 – 5.2 – 2.02 – Control (H + O + 53.8 – 2.77 – 4.8 – 1.93 – NIS) (F6) 73 36 6.11 120 0.6 87 0.23 88 (F10) 74.2 38 6.35 129 1.4 70 0.25 88 LSD (5%) 11.33 – 1.81 – 2.18 – 1.14 – Vica faba minor, faba bean Orobanche foetida Control (H) 64.8 – 5.39 – – – – – Control (H + IS) 65.4 – 5.65 – – – – – Control (H + F6) 89.4 37 9.41 66 – – – – Control (H + F10) 89 36 8.6 52 – – – – Control (H + O) 50.2 – 3.32 – 5.6 – 2.12 – Control (H + O + 54.2 – 3.41 – 4.4 – 2.28 – NIS) (F6) 73.4 35 6.08 79 1.4 68 0.43 81 (F10) 78.8 45 6.18 82 1 77 0.36 84 LSD (5%) 11.33 – 1.81 – 2.18 – 1.14 – H Faba bean only; H + NIS faba bean plus non-inoculated barley grains; H + O faba bean plus Orobanche; H + O + F Faba bean plus non- inoculated barley grains plus Orobanche.
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Table 3. Effect of isolates F6 and F10 on the number of tubercles and tubercle dry weight of Orobanche crenata and Orobanche foetida and on the height and dry weight of Faba bean, in non sterilized soil. Treatments Plant height Percent Plant dry Percent No. of Percent Tubercles dry Percent (cm) increase weight (g) increase tubercle reduction weight (g) reduction Vica faba minor, faba bean Orobanche crenata Control (H) 56.4 – 1.9 – – – – – Control (H + IS) 49.8 – 1.8 – – – – – Control (H + F6) 64 28 1 – – – – – Control (H + F10) 51.8 4 1.2 – – – – – Control (H + O) 39.8 – 0.8 – 2 – 0.025 – Control (H + O + 37.6 – 0.9 – 2,2 – 0.025 – NIS) (F6) 45.6 21 1.9 111 0 100 0 100 (F10) 45.4 20 1.6 77 0 100 0 100 LSD (5%) NS – NS – 0.59 – NS – Vica faba minor, faba bean Orobanche foetida Control (H) 56.4 – 1.9 – – – – – Control (H + IS) 49.8 – 1.8 – – – – – Control (H + F6) 64 28 1 – – – – – Control (H + F10) 51.8 4 1.2 – – – – – Control (H + O) 38.6 – 0.8 – 1.8 – 0.025 – Control (H + O + 30.4 – 0.6 – 2.6 – 0.025 – NIS) (F6) 48.4 59 1.6 166 0 100 0 100 (F10) 48.6 59 1 66 0 100 0 100 LSD (5%) NS – NS – 0.59 – NS – H Faba bean only; H + NIS faba bean plus non-inoculated barley grains; H + O faba bean plus Orobanche; H + O + F faba bean plus non inoculated barley grains plus Orobanche.
3,5 3 2,5 2 1,5 tubercles 1 0,5 Nomber of Orobanche 0 0 H+OF H+OC H+OF+F6 H+OC+F6 H+OF+ST H+OC+ST H+OF+F1 H+OC+F10
Figure 1. Pesta formulation effect on the number of Orobanche cre- nata and O. foetida tubercles. during a pot experiment or under field conditions. In The percentage of attachment of O. crenata to the Petri dishes the fungus reduced the germination of O. lentil host plant was also reduced by 79% as a result of crenata by 27% to 93% compared to the control. Tho- fungus inoculum. Bedi and Donchev (1991) suggested mas et al., (1999), found that Fusarium oxysporum f. that the black pigment of seeds protects the seed from sp. ortoceras colonized seeds of O. cumana, so the ger- fungal attack and they believed that the infection by the mination of inoculated seed was significantly reduced. fungus occurred after seed germination. Consequently, Zermane et al. (1999) found that the germination of the number of tubercles formation was significantly O. crenata was reduced by 50.3% when they used reduced by 98% in Petri dishes assays. Müller-Stöver F. oxysporum in root chamber assays. (2001) found that the mortality of O. aegyptiaca tu-
242 Evaluation of Fusarium as potential biological control against Orobanche on Faba bean in Tunisia bercles was significantly increased after inoculation Bedi J.S. and Donchev, N. (1991) Results on mycoherbicide in a root chamber. Accordingly, the same phenomena control of sunflower broomrape (Orobanche cumana) were also observed by Bouzoukov and Kouzmanova under glass house and field conditions. In: Fifth Interna- (1994). Thomas et al. (1998) suggested that F. oxyspo- tional Symposium on Parasitic Weeds. Nairobi, Kenya, rum inoculated in a root chamber, attacked germination pp. 76–82. Boari, A. and Vurro. M. (2004) Evaluation of Fusarium spp. and tubercles formation. Cohen et al. (2002) explained and other fungi as biological control agents of broomrapes the mortality process of tubercles; they suggested that (Orobanche ramose). Biological Control 30, 212–219. the hyphen penetrated the outer cells layer within 24 h, Bouzoukov, H. and Kouzmanova. I. (1994) Biological control reaching the centre of the tubercles by 48 h and infected of tobacco broomrape (Orobanche spp) by means of some nearly all cells by 72 h. Most of the infected tubercles fungi of the genus Fusarium. Biology and management of had died by 96 h. We observed necroses on inoculated Orobanche. In: Pieterse A.H., Verkleig, j.a.c. and Borg, tubercles and the same were observed by Linke et al. s. j. Ter. (eds) Proceedings of the Third International (1992). Workshop on Orobanche and Related Striga Research. Of the ten isolates tested, two F6 and F10 were the Royal Tropical Institute, Amsterdam, The Netherlands, most effective in reducing the percentage of tubercles pp. 532–538. in Petri dishes. Tests in with these two isolates in ste- Cohen, B.A., Amsellem, Z., Simcha, L.Y. and Gressel, J. (2002) Infection of tubercles of parasitic weed Orobanche rilized and non-sterilized soil showed that they could aegyptiaca by mycoherbicidal Fusarium species. 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