Biological Seed Treatment of Cereals with Fresh and Long-term Stored Formulations of Clonostachys rosea: Biocontrol Efficacy Against Fusarium culmorum

In six field experiments, seed treatment with Clonostachys rosea (IK726) significantly reduced disease caused by Fusarium culmorum. IK726 was active against the pathogen at average soil temperatures at sowing ranging from 6.2 to 12 °C. Both in the field experiments and in growth chamber experiments conducted in sand, dried and stored conidia of IK726 controlled F. culmorum as effectively as freshly harvested conidia. A high correlation was found between disease index ratings from field experiments and from corresponding growth chamber sand tests. Amendment with the stickers Pelgel or Sepiret did not influence control activity. The effective dosages of IK726 (cfu/seed) were estimated in bioassays and were very similar for freshly harvested conidia and for dried conidia. With a density of > 5×10³ conidia per seed more than 80% disease control was repeatedly obtained with both types of conidia.     

Biopriming of Infected Carrot Seed with an Antagonist, Clonostachys rosea, Selected for Control of Seedborne Alternaria spp

ABSTRACT An ecological approach was used to select fungal antagonists effective against the seedborne pathogens Alternaria dauci and A. radicina on carrot. Twenty-five and 105 isolates originating from cereal and carrot habitats were screened against the pathogens in planta, respectively. Irrespective of isolate origin, fungal isolates belonging to Clonostachys rosea controlled pre- and postemergence death caused by A. dauci and A. radicina as effectively as the fungicide iprodione. Isolate IK726 of C. rosea was used in biopriming a seed lot with 29% A. radicina and 11% A. dauci (highly infected), and a seed lot with 4% A. radicina and 7% A. dauci (low infection). Seeds were primed with water alone (hydropriming) or with addition of C. rosea IK726 (biopriming). The occurrence of A. radicina and A. dauci increased twofold and fivefold, respectively, during 14 days hydropriming, irrespective of the initial infection level. On highly infected seed, biopriming reduced the incidence of A. radicina to <2.3% and that of A. dauci to <4.8% while the level of both pathogens was <0.5% on bioprimed seed with a low initial infection rate. In sand stand establishment tests, hydroprimed seeds had a lower healthy seedling stand than nonprimed seeds, mainly due to a high degree of postemergence seedling death. In contrast, biopriming resulted in a seedling stand that was better than that of both nonprimed and hydroprimed seeds. C. rosea IK726 multiplied fivefold to eightfold, and microscopic observations using C. rosea IK726 transformed with a green fluorescent protein (GFP) reporter gene showed that seeds were covered with a fine web of sporulating mycelium of C. rosea. The positive effect of biopriming on healthy seedling stand remained after 5 months of storage at 4 degrees C and IK726 survived at high numbers on these seed. In this study, we demonstrated that bio-priming with the biocontrol strain C. rosea IK726 facilitates priming of infected seeds without risking adverse effects on seedling establishment.     

<Emphasis Type="Italic">In vivo</Emphasis> evaluation of essential oils and biocontrol agents combined with heat treatments on basil cv Genovese Gigante seeds against <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">basilici</Emphasis>

Seed treatments with essential oils (from savory and thyme) and biocontrol agents (Pseudomonas spp. and Fusarium oxysporum) have been evaluated in vivo after dry hot air treatments against Fusarium oxysporum f. sp. basilici on basil seeds. The savory and thyme essential oils showed a significant pathogen control activity because of their innate antifungal activity and because of the seed application method, but the dry hot pre-treatment did not show any obvious effect on the performance of the essential oil treatments. The dry heat treatment improved the Pseudomonas seed dressing effect against F.oxysporum f. sp. basilici, and showed important reductions in plant infection and the disease index on the treated seed plants, without any negative effect on seed germination. However, the pathogen control provided by the heat treatments combined with the application of the biocontrol agents never reached the same performance as the chemical treatments considered as the reference. Thus, short dry heat treatments on basil seeds have been shown to be a valid but complementary seed disinfection method against Fusarium wilt.     

Control of seed-borne pathogens on legumes by microbial and other alternative seed treatments

Greenhouse trials were carried out in order to test the efficacy of different seed treatments as alternatives to chemicals against Colletotrichum lindemuthianum cause of anthracnose on bean and Ascochyta spp. cause of Ascochyta blights on pea, respectively. Resistance inducers, commercially formulated microorganisms, non-formulated selected strains of different microorganisms (fungi, bacteria and yeasts) and plant extracts were applied as dry or liquid seed treatments on naturally infested seeds. Seedling emergence and disease incidence and/or severity were recorded. Almost all seed treatments turned out to be ineffective in controlling the Ascochyta infections, which is in line with the literature stating that these pathogens are difficult to control. The only alternative treatments that gave some control of Ascochyta spp. were thyme oil and a strain of Clonostachys rosea. The resistance inducers tested successfully controlled infections of bean by C. lindemuthianum. Among the formulated microorganisms, Bacillus subtilis-based formulations provided the best protection from anthracnose. Some strains of Pseudomonas putida, a disease-suppressive, saprophytic strain of Fusarium oxysporum and the mustard powder-based product Tillecur also proved to be effective against bean anthracnose. However, among the resistance inducers as well as among the other groups, certain agents caused a significant reduction of plant emergence. Different alternative seed treatments can therefore be used for the control of C. lindemuthianum on bean, while on pea only thyme oil and a strain of Clonostachys rosea showed some effectiveness against Ascochyta spp.     

Induction of defence related enzymes and phenolic compounds in lupin (Lupinus albus L.) and their effects on host resistance against Fusarium wilt

Two different biotic inducers [Pseudomonas fluorescens and Pseudomonas putida] and three different abiotic inducers [copper sulphate, indole butyric acid and potassium chloride] were tested for their efficacy in inducing resistance in lupin plants against Fusarium wilt disease caused by Fusarium oxysporum f. sp. lupini. Application of the biotic and abiotic inducers as seed treatments significantly reduced wilt disease incidence under greenhouse and field conditions. Potassium chloride and Pseudomonas fluorescens were superior. A time course of defence-related enzymes showed substantial increases in enzyme activities in induced infected seedlings compared with untreated healthy plants or infected controls. However, the magnitude of the increase varied among treatments. The maximum increases in chitinase and ??- glucanase activities were recorded at 12 and 8?days after inoculation with the pathogen, respectively. Also, the activity of phenylalanine ammonia lyase increased dramatically 8?days after inoculation. Greater accumulation of phenolic compounds and specific flavonoids upon infection with the pathogen was found in induced and/ or infected seedlings compared with healthy plants. In addition to inducing disease resistance, the treatments were accompanied by significant increases in crop parameters and seed yield compared with untreated controls.     

Isolation and selection of Hemileia Vastatrix antagonists

Organic coffee growing is rapidly increasing in Brazil, and many diseases, especially coffee leaf rust (Hemileia vastatrix), are threatening its production. This study is a first step towards a biocontrol program for coffee rust on organically grown plants. In three organic coffee farms in the state of Minas Gerais, 393 microbial strains including 154 bacterial and 239 fungal strains were isolated from leaves, leaf debris, and soil samples, and in 6 month-old coffee plants, 17 of these isolates reduced both the infection frequency (IF) and the number of H. vastatrix urediniospores produced per leaf (UPL) by more than 70 %. The isolates were identified as eight bacteria isolates, seven Bacillus spp. and one Pseudomonas sp., and nine fungal isolates, four Fusarium spp., two Penicillium spp., one Aspergillus sp., one Acremonium sp. and one Cladosporium sp. Each fungal and bacterial isolate was applied 0, 4, 8, 12 or 16 days before and 0, 4, 8, 12 or 16 days after H. vastatrix inoculation, and the efficiency in reducing both IF and UPL was evaluated. The efficiency was higher and lasted longer when the bacterial isolates were applied before H. vastatrix inoculation. Six Bacillus (B10, B25, B143, B157, B171, B175), two Fusarium (F205, F281), and one Pseudomonas (B286) isolates are potentially efficient as biocontrol agents of H. vastatrix and will be tested using field experiments.     

Increased bacterial endophyte populations in Nicotiana benthamiana with a branched‐chain alkane mixture

The aim of this study was to determine if treatment of soil with a branched‐chain alkane mixture known to induce resistance against Colletotrichum orbiculare also changes populations of bacterial endophytes from Nicotiana benthamiana. Eight culturable bacterial endophyte types matching six species of Bacillus and two species of Pseudomonas were found in roots, stem + petioles and/or leaves. Application of the branched‐chain alkane mixture resulted in significantly higher endophyte populations compared to the water or emulsifier controls for the Pseudomonas sp. LW3, Bacillus simplex LW4 and Bacillus pumilis LW5 colony types in roots and the B. simplex LW4 colony type in stem + petioles. The Pseudomonas sp. LW3 and B. simplex LW4 colony types also had higher populations in pure cultures under in vitro conditions with the branched‐chain alkane mixture compared to the controls. Inoculation with each of the eight colony types increased their population in the plant and induced resistance against C. orbiculare, with the most effective being Pseudomonas sp. LW3 and Pseudomonas alcaligenes SW1. Most of the endophytes could inhibit C. orbiculare growth in vitro, but the level of resistance in planta was not correlated to the ability of the colony type to inhibit C. orbiculare in culture. Thus, a branched‐chain alkane mixture can selectively affect the biomass of a subset of bacterial endophytes, demonstrating that it is a novel in planta endophyte growth promoter.     

Seed treatments associated with resistance inducers for management of <Emphasis Type="Italic">Pratylenchus brachyurus</Emphasis> in soybean

Pratylenchus brachyurus is one of the main limiting factors of soybean yield in Brazil, particularly because of inefficiency of the control methods when used individually. The present study aimed to assess the effect of associated methods, using seed treatment with nematicides (ST) and resistance inducers (RI), on nematode control in soybean plant in both field and greenhouse conditions. A field assay was conducted in an infested field and nematode population was assessed at sowing, 45, 75, and 100 days after sowing and the yield measured at end of crop cycle. The experiment was repeated in greenhouse. In another experiment, that was conducted in two different periods in a greenhouse, seed treatments and resistance inducers, alone or combined, were assessed under two initial populations of P. brachyurus (low IP = 500 specimens and high IP = 2000 specimens). The treatments did not reduce the number of nematodes g^?1 of root in field assay, but all seed treatments effectively controlled nematode population in the greenhouse assay. Most treatments reduced the number of nematodes g^?1 of root when nematode initial population was low (IP = 500) but when initial nematode population was high (IP = 2000) combinations of treatments which includes abamectin inhibited P. brachyurus reproduction. Chemical products did not affect yield but acibenzolar-S-methyl, alone or associated with other products, generally inhibit plant growth.     

Induced resistance in tomato plants against Fusarium wilt invoked by Fusarium oxysporum f.sp. dianthi

Tomato plants, susceptible toFusarium oxysporum f. sp.lycopersici, were inoculated by immersing the roots in a conidial suspension ofF. oxysporum f. sp.lycopersici race 1,F. oxysporum f. sp.dianthi race 2 or a mixture of both fungi. Plants inoculated withF. oxysporum f. sp.lycopersici showed disease symptoms after 2 weeks, whereas plants inoculated withF. oxysporum f. sp.dianthi or a mixture of both fungi remained symptomless for over 7 weeks, the duration of the experiment. In another experiment root systems of plants were split and each half was separately inoculated. One half was firstly inoculated withF. oxysporum f. sp.dianthi or treated with water, followed after a week by a second inoculation of the other half withF. oxysporum f. sp.lycopersici or by a water treatment. The disease symptoms in the half firstly inoculated withF. oxysporum f. sp.dianthi were significantly delayed, compared to plants of which that half had been treated with water. BecauseF. oxysporum f. sp.dianthi reduced disease symptoms caused byF. oxysporum f. sp.lycopersici without any direct interaction with this pathogen, it is concluded thatF. oxysporum f. sp.dianthi is able to induce resistance againstF. oxysporum f. sp.lycopersici in tomato plants.     

Effects of Host and Microbial Factors on Development of Clonostachys rosea and Control of Botrytis cinerea in Rose

Development of Clonostachys rosea in rose leaves and petals and control of Botrytis cinerea by the agent were investigated. C. rosea germinated, established endophytic growth, and sporulated abundantly whether the tissues were mature, senescent or dead when inoculated. Germination incidence was moderate on mature and senescent leaves (47% and 35%) and petals (31% and 43%), and high (>98%) on dead tissues. Sporulation of C. rosea in tissues inoculated when mature, senescent or dead averaged 41%, 61%, and 75% in leaves, and 48%, 87% and 53% in petals. When leaves were wounded with needles before inoculation, germination of C. rosea increased from 45–56% to 90–92%, but sporulation became high (>75%) regardless of wounds. When leaves were inoculated with C. rosea at 0–24h after wounding and subsequently with B. cinerea, germination of the pathogen was reduced by 25–41% and sporulation by 99%. A humid period prior to inoculation of senescent or dead leaves promoted communities of indigenous fungi, reduced sporulation of C. rosea and B. cinerea, and, in dead leaves, increased control of the pathogen associated with C. rosea. Applied at high density, isolates of indigenous Penicillium sp. and Alternaria alternata from rose interacted with C. rosea and reduced control of the pathogen by 16% and 21%, respectively. In conclusion, C. rosea markedly suppressed sporulation of B. cinerea in rose leaves and petals regardless of developmental stage, minor wounds, and natural densities of microflora. This versatility should allow C. rosea to effectively control inoculum production of B. cinerea in rose production systems.     

Non-chemical weed control on traffic islands: a comparison of the efficacy of five weed control techniques

The efficacy of five non‐chemical weed control methods for reducing weed cover on traffic islands was investigated in the growing season of 2004. Three trial sites were divided into six treatment areas which were treated with either flame, steam, hot air, hot water, brushes or left untreated. The treatments were carried out at regular intervals throughout the growing season. The percentage weed cover was measured every second week using a 75 × 75 cm quadratic frame with 100 squares. In the control areas, a rapid increase in weed cover was observed, whereas all treatments reduced weed cover. Hot water was the most effective method, although not significantly better than hot air or steam. Hot air treatment was more effective than brushing, whereas hot water was more effective than both flaming and brushing. The doses that were used were relatively high (150–355 kg ha^?1), partly because of the irregular shape of the traffic islands and the treatment intervals were quite short in comparison with those in similar studies. However, the treatments could keep down the weeds only to a certain extent. The present knowledge of the efficacy of various weed control methods, as well as an increase in our knowledge of adequate treatment intervals, supports an optimisation of hard‐surface weed control. Data and experience gained from these trials were used to develop further calibrated application studies.     

微生物源农药申嗪霉素的研制与应用

申嗪霉素是中国自主研发的一种新型微生物源农药,具有高效、安全、广谱等特点,其主要成分是甜瓜根际促生菌M18产生的次级代谢产物吩嗪-1-羧酸。文章重点就申嗪霉素产生菌M18的分离及其代谢产物的鉴定、申嗪霉素生物合成及调控机理最新研究进展、申嗪霉素高产工程菌株的构建和产业化、以及申嗪霉素大田防病试验结果及推广应用情况等进行详细综述,并对其抗菌作用机理进行探讨,旨在为申嗪霉素的生物合成机理研究、遗传和代谢改造以及推广应用提供参考。     

Potato blackleg: Epidemiology,host-pathogen interaction and control

Blackleg is caused byErwinia carotovora subsp.atroseptica (Eca) andE. chrysanthemi (Echr) in cool and hot climates respectively. The bacteria are opportunistic pathogens and rely on their strong pectolytic character to infect plants when conditions favor their multiplication. Blackleg is a seedborne disease and the bacteria can survive in a quiescent form in lenticels and wounds during storage. The contaminated mother tuber and not the blackleg plant is the main source of progeny tuber contamination. Other sources of the pathogen are airborne (insects and aerosols) erwinias deposited on leaves and from there to the soil and progeny tubers, and erwinias in rotting tubers smeared into wounds incurred during mechanical crop handling.Most seed tubers are contaminated but blackleg incidence is related to seed contamination level modulated by soil water status. Competitiveness of the erwinias in the rotting mother tuber is affected by temperature, Eca is favored at <25°C and Echr at higher temperatures. The ubiquitousE. carotovora subsp.carotovora apparently fails to compete successfully with the other erwinias and saprophytic pectolytic bacteria in mother tubers and therefore does not cause blackleg.Disease control measures are based on avoiding tuber contamination by cultural means (early harvesting), reducing tuber contamination level (dry storage and hot water treatment) and planting ‘clean’ seed identified by quantifying tuber contamination rather than by visual crop inspection. Finally, recently identified highly resistant, even under anaerobic conditions, wildSolanum spp. could be used to breed for resistant cultivars by conventional methods or by genetic engineering.     

Efficacy of different steam distribution systems against five soilborne pathogens under controlled laboratory conditions

The efficacy of three steam application techniques (steam injection, iron pan and sheet steaming) was evaluated against five soilborne pathogens under controlled laboratory conditions. Injection and pan steam systems proved to be efficient and feasible alternatives to traditional sheet steaming for suppressing Fusarium oxysporum f. sp. basilici at 60% moisture field capacity in sandy-loam soil. Injecting steam was the best technique to suppress F. oxysporum f. sp. basilici, F. oxysporum f. sp. raphani, F. oxysporum f. sp. conglutinans, Rhizoctonia solani and Phytophthora capsici. The mycelia of R. solani and P. capsici were very sensitive to heat and were effectively killed by injection of steam and by the pan steam system at 80% and 40% moisture field capacity.     

The effect of chemical and heat treatments on germination of Commelina benghalensis L. aerial seeds

The effects of concentrated sulphuric acid, dry heat, hot water and NaOCl treatments on the germinability of dormant large and small aerial Commelina benghalensis L. seeds were evaluated. Concentrated sulphuric acid and NaOCl treatments were more effective than the dry heat and hot water treatments in breaking the dormancy of C. benghalensis aerial seeds. Treatments increased germination of both seed types due to its effect on the seed coat integrity. A scanning electron microscope revealed that changes as a result of scarification occurred in the hilum region of the seed and in the seed coat surface. The large aerial seeds were affected by all treatments more than the small aerial seeds. Difference in germinability between the two seed types was related to the difference in their seed vigour.     

Effect of plant extracts and an essential oil on the control of brown spot disease, tillering, number of panicles and yield increase in rice

The effects of essential oils (EO), cold water (CWE), hot water (HWE) and ethanol (ETHE) extracts of Callistemon citrinus L. and Cymbopogon citratus (DC) Stapf on the radial growth of Alternaria padwickii (Ganguly) M.B. Ellis and Bipolaris oryzae (Breda de Haan) Shoemaker, the control of brown spot disease, the tillering, the number of panicles and the yield increase in rice were evaluated under laboratory and field conditions. In vitro, the growth of both fungi was completely inhibited by the EO of C. citrinus and C. citratus at 4,520 μg/ml and 452 μg/ml, respectively. For solvent extracts, the ETHE of C. citrinus was the most active and inhibited 80–85 % of the fungal growth followed by the CWE of C. citratus with 77 % and 36 % diameter reduction against B. oryzae and A. padwickii, respectively at 10 000 μg/ml. Under laboratory conditions, seed treatment with the EO of C. citrinus reduced the incidence of B. oryzae in seeds by 85–100 % compared to the non-treated controls. Similarly, the seed treatment increased the germination of an irrigated rice cultivar by 10.6 %, whereas the percentage of germinated seeds of upland rice was not significantly affected. The highest germination (85–94 %) was found in the non-treated and treated samples with a low incidence (0–4 %) of B. oryzae. Under field conditions, the combined use of the essential oil of C. citrinus as a seed treatment and spraying the plants with 2 % ethanol followed by 2 % (w/v) aqueous extracts of C. citrinus or C. citratus increased the emergence, tillering, panicles/plant and the grain yield by 25–55 % of the irrigated rice. In addition, the brown spot severity was reduced by 36–42 %. For the upland rice, the treatments led to similar results with the grain yield increase of 54–137 % and 20–80 % reduction in the brown spot severity. From our results, we concluded that the EO and solvent extracts of C. citrinus and C. citratus have potential as control agents against brown spot and other seed-borne fungal diseases in rice under both conventional and organic farming.     

Improving control of crown rot disease and quality of pesticide-free banana fruit by combining Bacillus amyloliquefaciens DGA14 and hot water treatment

The present study aimed at improving the control of crown rot disease and quality of pesticide-free banana banana fruit by integrated approach combining Bacillus amyloliquefaciens DGA14 and hot water treatment (HWT). The efficacy of treatment combination was tested on cultures of crown rot-causing pathogens, Lasiodiplodia theobromae, Thielaviopsis paradoxa, Colletotrichum musae and Fusarium verticillioides, and postharvest application. Combination of DGA14 and HWT inhibited mycelium growth by 82.61 % as compared to 66.68 % by DGA14 and 37.90 % by HWT, as single treatments. Spore germination was totally inhibited in DGA14 with or without HWT. Postharvest application with DGA14 + HWT gave 90 % control of fruit decay comparable to 93 % of synthetic fungicide. Fruit treated alone with hot water or in combination with DGA14 incurred the lowest loss in weight. The over-all quality of fruit with DGA14 + HWT was better appearance and firmer texture which was statistically comparable with fungicide-treated banana. DGA14 + HWT was more consistent in crown rot control i.e., limited variation in severity of disease within a treatment as compared to DGA14 and HWT, as stand-alone treatments.     

Flower infection of Brassica oleracea with Xanthomonas campestris pv. campestris results in high levels of seed infection

During seed production, Brassica seed may become infected with Xanthomonas campestris pv. campestris after systemic colonization of plants upon leaf infection, or alternatively, after flower infection. Polytunnel experiments were conducted in 2007 and 2008 to study the relative importance of these colonization routes resulting in seed infection. Cauliflower plants (Brassica oleracea) were spray-inoculated at the 8-leaf stage, after formation of cauliflowers or during flowering, at which stage leaves or blossoms were inoculated. Inoculation at all stages resulted in a relatively high percentage of systemic infection; the average estimated infection incidences for stem base and peduncle infections were 16 % and 19 %, respectively. When seed samples were examined by dilution plating for deep-seated infection following hot water treatment, Xcc was detected in 61 % of the 23 seed samples harvested from plants with inoculated flowers. However, symptom development in seedlings raised from the seeds could not be confirmed in a grow-out test under favourable conditions for Xcc infection at a high RH (>95 %) and a relative high temperature (28 °C). Xcc was not detected in 59 seed samples harvested from leaf-inoculated plants with the exception of one sample from plants inoculated at peduncle formation. In a third polytunnel experiment carried out in 2009, the population dynamics of Xcc on inoculated flowers was investigated. Following spray-inoculation of flowers, 52 % of the flowers were infected with Xcc. During development of siliques, infection incidence decreased slowly and at 56 dpi, 20 % of the superficially disinfected siliques were infected with Xcc. It was estimated that 0.18 % of the seeds was infected and that 1–10 % of the infected siliques contained infected seeds. The implications of these results for control of Xcc in a seed production crop are discussed.     

Blind seed disease of ryegrass in the Netherlands

The blind seed disease of ryegrass, caused by the fungusGloeotinia temulenta, is always present but seldom becoming important in the Netherlands. In 1965, however, probably due to a very rainy summer, it developed epiphytotically especially in the awned ryegrasses, Italian and Westerwold. With the waterdrop method an average seed infection of 19.2% was observed. Westerwold ryegrass was more strongly infected than Italian, and tetraploids heavier than diploids. The disease was most prevalent in the coastal provinces, petering out towards the South-East. In samples of the 1964 and earlier harvests the fungus was also found, although it seldom reached high percentages. The relationship between infection percentage and germinating capacity percentage of the samples was approximately a 45° one, the correlation coefficient between both values being 0.77. Extra cleaning of seed samples did not influence their infection percentages. In experiments on seed treatment the agar method was used for determining viable infection. The hot water method with 30 minutes at 50°C as indicated by Irish workers, as well as the mercurial treatment in combination with heat advocated in Scotland, appeared to leave part of the infection surviving. A warm water treatment with 2–2 1/2 hours at 45°–46°C, however, proved to be very satisfactory by totally eradicating the infection without injuring the seed.