Genetic approaches to sustainable pest management in sugar beet (Beta vulgaris)

Sugar beet (Beta vulgaris) is an important arable crop, traditionally used for sugar extraction, but more recently, for biofuel production. A wide range of pests, including beet cyst nematode (Heterodera schachtii), root‐knot nematodes (Meloidogyne spp.), green peach aphids (Myzus persicae) and beet root maggot (Tetanops myopaeformis), infest the roots or leaves of sugar beet, which leads to yield loss directly or through transmission of beet pathogens such as viruses. Conventional pest control approaches based on chemical application have led to high economic costs. Development of pest‐resistant sugar beet varieties could play an important role towards sustainable crop production while minimising environmental impact. Intensive Beta germplasm screening has been fruitful, and genetic lines resistant to nematodes, aphids and root maggot have been identified and integrated into sugar beet breeding programmes. A small number of genes responding to pest attack have been cloned from sugar beet and wild Beta species. This trend will continue towards a detailed understanding of the molecular mechanism of insect–host plant interactions and host resistance. Molecular biotechnological techniques have shown promise in developing transgenic pest resistance varieties at an accelerated speed with high accuracy. The use of transgenic technology is discussed with regard to biodiversity and food safety.     

The influence of colonizing methylobacteria on morphogenesis and resistance of sugar beet and white cabbage plants to <Emphasis Type="Italic">Erwinia carotovora</Emphasis>

The influence of colonization of sugar beet (Beta vulgaris var. saccharifera (Alef) Krass) and white cabbage (Brassica oleracea var. capitata L.) plants by methylotrophic bacteria Methylovorus mays on the growth, rooting, and plant resistance to phytopathogen bacteria Erwinia carotovora was investigated. The colonization by methylobacteria led to their steady association with the plants which had increased growth speed, root formation and photosynthetic activity. The colonized plants had increased resistance to Erwinia carotovora phytopathogen and were better adapted to greenhouse conditions. The obtained results showed the perspectives for the practical implementation of methylobacteria in the ecologically clean microbiology substances used as the plant growth stimulators and for the plant protection from pathogens.     

Suppression of Fusarium oxysporum with recombinant polygalacturonase inhibiting proteins (BvPGIPs) extracted from sugar beet roots

Soil-borne fungus Fusarium oxysporum f. sp. betae (Fob) is the causative agent of Fusarium yellows in sugar beet. Leaf interveinal yellowing and root vascular discoloration significantly reduce root yield as well as sucrose content and juice purity. Fob, like other fungal pathogens, initiates disease development by secreting polygalacturonase (PG) enzymes to break down plant cell walls during early stages of infection. To protect themselves, plants produce polygalacturonase-inhibiting proteins (PGIPs). In our study of sugar beet root defense responses, several PGIP genes (BvPGIPs) were identified. To determine if BvPGIPs inhibit Fob PGs, genes BvPGIP1, BvPGIP2 and Bv(FC607)PGIP1 were fused with the CaMV 35S promoter and each was expressed individually in sugar beet hairy roots. We demonstrate that all three recombinant BvPGIP proteins inhibited Fob and F. oxysporum f. sp. gladioli (Fog) PGs. A comparable level of BvPGIP activity was observed against Fob PGs, while BvPGIP2 showed higher activity against Fog PGs. Similar results were obtained when recombinant PGIPs were used to bioassay effects on Fob and Fog spore germination and hyphal growth. This is a first report that documents F. oxysporum inhibition by overexpressing BvPGIPs that may lead to improved Fusarium yellows resistance in sugar beet.

     

Response of subterranean clover cultivars to root rot fungi

Twelve widely grown cultivars of subterranean clover (Trifolium subterraneum) were screened both under controlled environment conditions for their resistance to five fungi commonly associated with root rot and under field conditions for their resistance to natural root infections. All cultivars showed decreased seedling survival (particularly from Pythium irregulare and Rhizoctonia solani), tap and lateral root rot (particularly from Fusarium avenaceum, P. irregulare, and R. solani) and reduced plant size (particularly from R. solani and P. irregulare). Individual cultivars generally differed in their response to the five pathogens and for any one pathogen there was generally a range of cultivar susceptibilities. Cultivars with the best resistance to individual root pathogens were identified. The results for the five individual pathogens under controlled conditions only showed correlation with field data for some of the parameters compared.     

Evaluation of five sugar beet varieties for root-knot nematode and root-rot fungal infection

Survey results during 2010–2011 season revealed that the Meloidogyne spp., Helico–tylenchus spp. and Tylenchorhynchus spp. were the common plant parasitic nematodes in the rhizospheres of sugar beet in El-Behera, El-Fayoum and Beni Sueif Governorates. Aspergillus spp., Aspergillus niger, Fusarium spp., Penicillium spp., Penicillium chrysogenum, Penicillium citrinum, Rhizoctonia spp., Rhizopus nigricans, Trichoderma spp. and others were also the common fungi in the same rhizospheres. The rhizosphere of El-Behera Governorate was highly infected with Meloidogyne spp., Fusarium spp. and Rhizoctonia spp., compared to the rhizospheres of El-Fayoum and Beni Sueif, respectively. Evaluation of five of sugar beet cultivars, viz. Chems, Dema-Poly, DS 9001, Manila and Ras-Poly to infection, with Meloidogyne incognita (root-knot nematode), Fusarium solani and Rhizoctonia solani (root rot disease) was carried out under naturally field infection condition in the Nubariya region, Behera Governorate during 2011–2012 season. Host susceptibility rating revealed that the varieties of Ras-Poly, DS 9001 and Manila can be considered as susceptible, while the varieties of Dema-Poly and Chems can be considered as highly susceptible. Pathological results showed that the varieties of Dema-Poly and Ras-Poly were highly infected with F. solani, while not infected with R. solani. The varieties of DS 9001, Manila and Chems were moderately infected with the two pathogens.     

Sugar Beet-Associated Bacterial and Fungal Communities Show a High Indigenous Antagonistic Potential Against Plant Pathogens

The aim of this study was to analyze microbial communities in/on sugar beet with special focus on antagonists toward plant pathogens. For this purpose, the composition of microorganisms isolated from the rhizosphere, phyllosphere, endorhiza, and endosphere of field-grown sugar beet plants was analyzed by a multiphasic approach at three different plant development stages at six locations in Europe. The analysis of microbial communities by Single Strand Conformation Polymorphism (SSCP) of 16S/18S rRNA clearly revealed the existence of discrete microenvironment- and site-specific patterns. A total of 1952 bacterial and 1344 fungal isolates screened by dual testing for antagonism toward the pathogens Aphanomyces cochlioides, Phoma betae, Pythium ultimum, and Rhizoctonia solani resulted in 885 bacterial (=45%) and 437 fungal (=33%) antagonists. In general, the indigenous antagonistic potential was very high and influenced by (a) the location, (b) the plant developmental stage, and (3) the microenvironment. Furthermore, we showed for the first time that the antagonistic potential was highly specific for each target pathogen. The majority of antagonistic microorganisms suppressed only one pathogen (bacteria: 664 = 75%; fungi: 256 = 59%), whereas the minority showed a broad host range (bacteria: 4 = 0.5%; fungi: 7 = 1.6%). The bacterial communities harbored the highest antagonistic potential against P. ultimum, whereas the fungal communities contained more antagonists against A. cochlioides and R. solani. In contrast to their high proportion, only a low diversity of antagonists at genotypic and species level was found. Novel antagonistic species, e.g., Subtercola pratensis or Microbacterium testaceum were found in the internal part of the sugar beet body.     

Sporamin-mediated resistance to beet cyst nematodes (Heterodera schachtii Schm.) is dependent on trypsin inhibitory activity in sugar beet (Beta vulgaris L.) hairy roots

Sporamin, a sweet potato tuberous storage protein, is a Kunitz-type trypsin inhibitor. Its capability of conferring insect-resistance on transgenic tobacco and cauliflower has been confirmed. To test its potential as an anti-feedant for the beet cyst nematode (Heterodera schachtii Schm.), the sporamin gene SpTI-1 was introduced into sugar beet (Beta vulgaris L.) by Agrobacterium rhizogenes-mediated transformation. Twelve different hairy root clones expressing sporamin were selected for studying nematode development. Of these, 8 hairy root clones were found to show significant efficiency in inhibiting the growth and development of the female nematodes whereas 4 root clones did not show any inhibitory effects even though the SpTI-1 gene was regularly expressed in all of the tested hairy roots as revealed by northern and western analyses. Inhibition of nematode development correlated with trypsin inhibitor activity but not with the amount of sporamin expressed in hairy roots. These data demonstrate that the trypsin inhibitor activity is the critical factor for inhibiting growth and development of cyst nematodes in sugar beet hairy roots expressing the sporamin gene. Hence, the sweet potato sporamin can be used as a new and effective anti-feedant for controlling cyst nematodes offering an alternative strategy for establishing nematode resistance in crops.     

Physiological characterization of opine-utilizing rhizobacteria for traits related to plant growth-promoting activity

Thirty-two strains of opine-utilizing rhizobacteria were evaluated for physiological traits which have been related to plant growth-promoting activity. Tests included antibiosis against two bacterial and eight fungal pathogens of potato (Solanum tuberosum L.), production of hydrogen cyanide and fluorescent pigment production. On average, 71 and 12% of the bacteria inhibited the growth of Erwinia carotovora subsp. carotovora and Agrobacterium tumefaciens, respectively. The growth of Botrytis sp. was inhibited by 62% of the bacteria, and half of these produced an inhibition zone of more than 7 mm in diameter. Fusarium solani, Colletotrichum coccodes, Phoma exigua, Verticillium dahliae, F. oxysporum, V. albo-atrum and F. sambucinum were antagonized by 43, 34, 31, 25, 19, 18, and 12% of the bacteria, respectively. Only four strains produce hydrogen cyanide. The inhibition of a plant pathogen was not correlated to the production of fluorescent pigment. No strain produced a hypersensitive reaction whereas only three strains induced soft-rot and two produced polygalacturonase. Some opine-utilizing rhizobacteria were strong inhibitors of all plant pathogens, while most were active against specific plant pathogens.     

Influence of Sugar Beet Seed Treatment with Pseudomonas fluorescens and Low Fungicide Doses on Infection with Pythium and Plant Yield and Quality

The effect of sugar beet seed inoculation with the bacterium Pseudomonas fluorescens and treatment with the fungicides Thiram 42‐S and Dithane S‐60 with and without seed inoculation aiming to control the root decay agents Pythium ultimum and Pythium debarianum was studied during a 2‐year trial on two soil types (Mollic Gleysols and Eutric Cambisols). The influence of the treatments on parameters of sugar beet yield and quality such as root yield, sugar content, sugar in molasses, sugar yield as well as percentage of the infected and decayed plants as a consequence of parasitic oomycete infestation will be described.     

Chemical control of beet cyst-nematode, Heterodera schachtii, in some peaty loam soils

In peaty loam soils, aldicarb or oxamyl mixed with the top 15 cm of the soil in spring before sugar beet seeds were sown, minimised invasion of the roots by larvae of the beet cyst-nematode, Heterodera schachtii, so preventing injury to the seedlings, and greatly increased sugar yields in heavily infested soil. Small amounts of both compounds were often as effective as larger amounts. Nematode increase on sugar beet roots was slow. Aldicarb or oxamyl lessened nematode increase in four years out of five. Fumigating predetermined row positions with dichloropropene mixtures (D-D, Telone) or incorporating aldicarb or methomyl shallowly in soil, later occupied by the roots of sugar beet seedlings, did not control the nematode, although sugar yields were sometimes increased.     

Grape marc compost: microbial studies and suppression of soil-borne mycosis in vegetable seedlings

Compost suppression of soil-borne diseases in horticultural crops has been attributed to the activities of antagonistic microorganisms. A great diversity of microorganisms, capable of suppressing pathogens naturally colonize compost. A large number of microbes appeared in microbiological analyses of grape marc compost. Most microorganisms were bacteria. Average percentages were 31% mesophilic and 28% thermophylic bacteria, 16% mesophilic actinomycetes and 20% thermophylic actinomycetes. Only a few mould and yeast morphologies were obtained, 4% and 1% respectively. Antagonist in vitro assays were performed with 432 microbial morphologies isolated from grape marc compost. The microbes isolated were extremely effective antagonists in in vitro assays against all the fungal pathogens tested. Seven microorganisms were selected for further bioassay with Rhizoctonia solani in radish, Fusarium oxysporum f. sp. radicis-cucumerinum in melon, and Phytophthora parasitica in tomato and two microorganisms with Pythium aphanidermatum in cucumber. Those experiments indicate that grape marc compost reduces the severity of Pythium damping-off in cucumber, but does not reduce the severity of Phytophthora root rot in tomato, Fusarium oxysporum f. sp. radicis-cucumerinum in melon and Rhizoctonia solani in radish. Better suppressive effects were not demonstrated by either compost or vermiculite amended with microbes selected from grape marc compost.     

Minor root rot pathogens of cassava (Manihot esculenta Crantz) in Nigeria

Many different species of fungi are often isolated from rotted cassava root tubers and pathogenicity studies have often implicated Botryodiplodia theobromae and Fusarium solani as the major causal pathogens. Consequently, more attention has often been focused on Botryodiplodia theobromae and Fusarium solani with little or no attention on the other minor pathogens. Considering the increasing importance of cassava to the Nigerian economy and the fact that minor root rot pathogens of cassava today could become major tomorrow, the aim of this research is to determine the incidence, pathogenicity and symptoms of the minor root rot pathogens in cassava from cassava fields within the derived savanna and the humid forest of Nigeria. Isolation of associated fungi was done on rotted root samples and the pathogenicity of these isolates were established by inoculating them into healthy cassava tuberous roots and subsequently reisolating them from resulting rotted tissue. The less frequently isolated fungi where Macrophomina sp., Trichoderma sp., Aspergillus niger, Aspergillus flavus, Sclerotium rolfsii and Fungus ‘A’ (a yet to be identified fungus). Repeated experiments confirmed a constant relationship between inoculated fungus and the resulting rotted tissue colour. The root rot tissue colours associated with inoculated pathogens in the laboratory were identical with the pathogens colony colour on potato dextrose agar.     

Suppression of sugar beet damping-off caused by Rhizoctonia solani using bacterial and fungal antagonists

Rhizoctonia damping-off caused by Rhizoctonia solani Kühn, is one of the most damaging sugar beet diseases. It causes serious economic damage wherever sugar beets are grown. Biological control is an efficient and environmentally friendly way to prevent damping-off disease. Suppression of damping-off disease caused by R. solani was carried out by four isolates of Bacillus subtilis (Ehrenberg) Cohn as well as three isolates of each of Trichoderma harzianum Rifai and Trichoderma hamatum (Bonord.) Bainier. The effect of Bacillus and Trichoderma isolates against R. solani was investigated in vitro and tested on sugar beet plants under greenhouse conditions. Isolates of Bacillus and Trichoderma were able to inhibit the growth of R. solani in dual culture. Furthermore, Trichoderma isolates gave high antagonistic effect than isolates of B. subtilis. Under greenhouse conditions, coating seeds by T. harzianum and B. subtilis separately, reduced seedling damping-off significantly. However, applications of T. harzianum increased the percentage of surviving plants more than B. subtilis in comparison to control. The obtained results indicate that T. harzianum and B. subtilis are very effective biocontrol agents that offer potential benefit in sugar beet damping-off and should be harnessed for further biocontrol applications.     

Biocontrol activity of salt tolerant Streptomyces isolates against phytopathogens causing root rot of sugar beet

Biological control of fungi causing root rot on sugar beet by native Streptomyces isolates (C and S2) was evaluated in this study. The dry weight and colony forming unit (CFU) of S2 and C increased when 300 mM NaCl was added to medium. The in vitro antagonism assays showed that both isolates had inhibitory effect against Rhizoctonia solani AG-2, Fusarium solani and Phytophthora drechsleri. In dual culture, Streptomyces isolate C inhibited mycelial growth of R. solani, F. solani and P. drechsleri 45%, 53% and 26%, respectively. NaCl treatment of medium increased biocontrol activity of soluble and volatile compounds of isolate C and S2. After salt treatment, growth inhibition of R. solani, F. solani and P. drechsleri by isolate C increased up to 59%, 70% and 79%, respectively. To elucidate the mode of antagonism, protease, chitinase, beta glucanase, cellulase, lipase and α-amylase activity and siderophore and salicylic acid (SA) production were evaluated. Both isolates showed protease, chitinase and α-amylase activity. Also, biosynthesis of siderophore was detectable for both isolates. Production of siderophore and activity of protease and α-amylase increased after adding salt for both isolates. In contrast, chitinase activity decreased significantly. Production of SA, beta glucanase and lipase by isolate S2 and biosynthesis of cellulase by isolate C were observed in presence and absence of NaCl. Soil treatment with Streptomyces isolate C inhibited root rot of sugar beet caused by P. drechsleri, R. solani and F. solani. Results of this study showed that these two Streptomyces isolates had potential to be utilized as biocontrol agent against fungal diseases especially in saline soils.     

林下参内生球毛壳菌FS-01对人参病原真菌的抑制作用

为探究林下参内生真菌球毛壳菌(Chaetomium globosum)FS-01菌株对人参病原菌的抑菌作用,该研究在实验室条件下,测定了FS-01菌株菌丝、发酵液和孢子悬浮液对人参黑斑病菌(Alternaria panax)、人参菌核病菌(Sclerotinia schinseng)、人参灰霉病菌(Botrytis cinerea)、人参立枯病菌(Rhizoctonia solani)、人参根腐病菌(Fusarium solani)5种人参病原菌的抑制作用。结果表明:内生真菌球毛壳菌FS-01对5种病原菌均有抑制作用,其中,对人参黑斑病菌的抑制作用最高,为30.80%,其次是人参立枯病菌、人参菌核病菌、人参根腐病菌和人参灰霉病菌; 发酵液抑菌实验结果表明,在加入内生真菌球毛壳菌FS-01菌株发酵液的PDA培养基上,对人参灰霉病菌的抑制作用最高,为82.09%,其次是人参菌核病菌、人参黑斑病菌、人参立枯病菌和人参根腐病菌; 孢子抑菌实验结果表明,在加入内生真菌球毛壳菌FS-01菌株孢子悬浮液的PDA培养基上,对人参黑斑病菌的抑制作用最高,为83.72%,其次是人参灰霉病菌、人参立枯病菌、人参菌核病菌和人参根腐病菌。综上结果认为,内生真菌球毛壳菌FS-01菌株对人参病原菌均有很高的抑菌作用,可作为人参病原菌的生防菌株资源。     

Populations of Rhizoctonia solani in soil under crops in rotation with sugar beet

Using a soil debris isolation method, populations of Rhizoctonia solani were monitored over a 4 -yr period in four fields which were initially cropped to sugar beet and in which four areas of Rhizoctonia crown rot diseased beets (DA) and four areas of apparently healthy beets (AH) had been selected and precisely located. Soil from these areas was assayed during the subsequent crops, which included sugar beet, tomato, cucumber, maize and soybean. No significant differences in colony counts were found between the soils in DA and AH on any of 30 sampling dates. R. solani population counts were, in general, quite low, except under sugar beet and following tomato harvest. Areas of diseased beet and high R. solani soil populations that developed in subsequent sugar beet crops did not necessarily coincide with the previously selected diseased areas. High R. solani populations developed from parasitic activity on sugar beet or saprophytically on tomato crop residues. Of the other crops, both maize and soybean may have slightly increased the low R. solani residual populations in soil. The monitoring of R. solani populations in the season prior to, and during the early season of sugar beet cropping did not provide a basis for forecasting disease in fields or sites within fields. The initiation of disease patches in these sugar beet fields was therefore governed by factors other than inoculum density.     

Characterization of Fusarium oxysporum Isolates from Common Bean and Sugar Beet Using Pathogenicity Assays and Random-amplified Polymorphic DNA Markers

Fusarium wilt is an economically important fungal disease of common bean and sugar beet in the Central High Plains (CHP) region of the USA, with yield losses approaching 30% under appropriate environmental conditions. The objective of this study was to characterize genetic diversity and pathogenicity of isolates of Fusarium oxysporum obtained from common bean and sugar beet plants in the CHP that exhibited Fusarium wilt symptoms. A total of 166 isolates of F. oxysporum isolated from diseased common bean plants were screened for pathogenicity on the universal susceptible common bean cultivar ‘UI 114’. Only four of 166 isolates were pathogenic and were designated F. oxysporum f.sp. phaseoli (Fop). A set of 34 isolates, including pathogenic Fop, F. oxysporum f.sp. betae (Fob) isolates pathogenic on sugar beet, and non‐pathogenic (Fo) isolates, were selected for random‐amplified polymorphic DNA (RAPD) analysis. A total of 12 RAPD primers, which generated 105 polymorphic bands, were used to construct an unweighted paired group method with arithmetic averages dendrogram based on Jaccard's coefficient of similarity. All CHP Fop isolates had identical RAPD banding patterns, suggesting low genetic diversity for Fop in this region. CHP Fob isolates showed a greater degree of diversity, but in general clustered together in a grouping distinct from Fop isolates. As RAPD markers revealed such a high level of genetic diversity across all isolates examined, we conclude that RAPD markers had only limited usefulness in correlating pathogenicity among the isolates and races in this study.     

A simple and rapid technique for fluorescence staining of fungal nuclei

A technique for staining fungal nuclei using fluorescence stain Hoechst Dye 33258 in McIlvaine standard buffer of pH 7.26–7.44 is reported. It is a broad-spectrum fungal nuclear staining tool found to be effective onAgaricus bisporus, Alternaria helianthi, Fusarium oxysporum f. sp.lini, Penicillium binellum, Pythium ultimum, Rhizoctonia solani, andSaccharomyces cerevisiae. Conidial nuclei ofAlternaria helianthi, Fusarium oxysporum f. sp.lini, andPenicillium binellum also stained well.     

Citrus replant problem in Iraq III. Interactive effect of soil fungi and allelopathy

The interactive effects of citrus root residues and soil fungi on citrus replant problems were investigated. The results indicated that incorporation of citrus root residues in combination with the pathogenic fungiPhytophthora citrophthora, Pythium aphanidermatum andFusarium solani in soil caused more reduction to sour orange growth than did the root residues alone. Subsequent experiments showed that extracts of different parts of sour orange and leachates of some soil fungi increased the disease index of citrus roots grownin vitro. The citrus extracts did not affect growth of the test fungi.Thus, it appears that allelopathic compounds of plant and microbial origins build up in old citrus soil and may act as predisposal agents for the infection of citrus roots by soil pathogens.     

Biocontrol of fungal root rot diseases of crop plants by the use of rhizobia and bradyrhizobia

Twenty-oneRhizobium andBradyrhizobium strains were testedin vitro against the mycelial growth of three pathogenic fungi on solid and liquid media. All tested rhizobia and bradyrhizobia significantly suppressed the growth of the three soil-borne root-infecting fungi (Fusarium solani, Macrophominia phasolina andRhizoctonia solani) either in the absence or presence of iron. This indicates that the siderophore played a minor role in the biocontrol potential ofRhizobium andBradyrhizobium against pathogenic fungi. Pot experiments revealed that the numbers of propagules causing disease after 4 weeks of planting varied with species and host plant. The three most activeRhizobium andBradyrhizobium strains (R. leguminosarum bv.phaseoli TAL 182,B. japonicum TAL 377 andBradyrhizobium sp. (lupin) WPBS 3211 D) tested under greenhouse conditions for their ability to protect one leguminous (soybean) and two non-leguminous (sunflower and okra) seedlings from root rot caused byFusarium solani, Macrophominia phaseolina andRhizoctonia solani provided significant suppression of disease severity compared with nonbacterized control in both leguminous and non-leguminous seedlings.Bradyrhizobium sp. (lupin) WPBS 3211 D provided the lowest degree of resistance against all the tested pathogens with all host plants. *** DIRECT SUPPORT *** A00EN058 00013