Kathy S. Lawrence

Integrated signaling networks in plant responses to sedentary endoparasitic nematodes: a perspective.

Sedentary plant endoparasitic nematodes can cause detrimental yield losses in crop plants making the study of detailed cellular, molecular, and whole plant responses to them a subject of importance. In response to invading nematodes and nematode-secreted effectors, plant susceptibility/resistance is mainly determined by the coordination of different signaling pathways including specific plant resistance genes or proteins, plant hormone synthesis and signaling pathways, as well as reactive oxygen signals that are generated in response to nematode attack. Crosstalk between various nematode resistance-related elements can be seen as an integrated signaling network regulated by transcription factors and small RNAs at the transcriptional, posttranscriptional, and/or translational levels. Ultimately, the outcome of this highly controlled signaling network determines the host plant susceptibility/resistance to nematodes.

Biocontrol of the Reniform Nematode by Bacillus firmus GB-126 and Paecilomyces lilacinus 251 on Cotton

Due to increased restrictions on the use of chemical nematicides, alternative nematode management strategies, including biocontrol, are needed. The objectives of this study were to evaluate the potential of Bacillus firmus GB-126 and Paecilomyces lilacinus 251 in commercial formulations applied separately or concomitantly to manage Rotylenchulus reniformis in cotton grown under greenhouse, microplot, and field conditions. In the greenhouse, seed treated with B. firmus (1.4 × 107 CFU/seed), an application of P. lilacinus (0.3% vol/vol of water), or the combination of B. firmus and P. lilacinus reduced the number of females, eggs, and vermiforms of R. reniformis (P ≤ 0.02) and increased populations of free-living nematodes (P ≤ 0.01). In microplots and field conditions, populations of R. reniformis vermiforms decreased when exposed to B. firmus and P. lilacinus biocontrol agents at midseason (P ≤ 0.04). Furthermore, stem diameter and free-living nematode numbers increased (P ≤ 0.01) with the combination of B. firmus and P. lilacinus. In the field, numbers of females, eggs, and vermiform life stages at the end of the growing season decreased in the presence of the biocontrol agents applied individually or concomitantly (P ≤ 0.01). Cotton yields from the application of B. firmus GB-126 and P. lilacinus 251 were similar to those from aldicarb, the chemical nematicide standard.

Efficacy of Bacillus subtilis MBI 600 Against Sheath Blight Caused by Rhizoctonia solani and on Growth and Yield of Rice

Rice sheath blight disease (ShB), caused by Rhizoctonia solani, gives rise to significant grain yield losses. The present study evaluated the efficacy of Integral®, the commercial liquid formulation of Bacillus subtilis strain MBI 600, against rice ShB and for plant growth promotion. In greenhouse studies, four log concentrations of Integral (from 2.2×106 to 2.2×109 cfu/mL) were used as seed treatment (ST). After 25 d, seedlings were dipped (SD) into Integral prior to transplanting. At 30 d after transplanting (DAT), leaf sheaths were inoculated with immature sclerotia of the pathogen. At 45 DAT, a foliar spray (FS) with Integral was applied to some treatments. The fungicide control was 50% carbendazim at 1.0 g/L, and a nontreated control was also included. Overall, there were 10 treatments, each with five replications. ShB severity was rated at 52 DAT, and seedling height and number of tillers per plant were rated at 60 DAT. In 2009, two field trials evaluated Integral at 2.2×108 and 2.2×109 cfu/mL. Integral was applied as ST, and seedlings were produced in a nursery bed. After 32 d, seedlings were treated with Integral as SD and transplanted into 10 m2 blocks. Foliar sprays were given at 45 and 60 DAT. There were seven treatments, each with eight replications arranged as a factorial randomized complete block design. At 20 DAT, the plots were broadcast inoculated with R. solani produced on rice grains. Seedling height before transplanting, ShB severity at 90 DAT, and grain yield at harvest were recorded. Integral at 2.2×109 cfu/mL provided significant increase of seedling heights over other treatments under greenhouse conditions. The Integral treatments of ST + SD + FS at 2.2×109 cfu/mL significantly suppressed ShB over other treatments. In field studies, Integral provided significant increase of seedling height in nursery, and number of tillers per plant, compared with the control. ShB severity was significantly suppressed with higher concentrations of Integral compared to lower concentrations. Grain yield were the highest at an Integral concentration of 2.2×109 cfu/mL. Overall, Integral significantly reduced ShB severity, enhanced seedling growth, number of tillers per plant and grain yield as ST + SD + FS at the concentration of 2.2×109 cfu/mL under the conditions evaluated.

Biological Control of Meloidogyne incognita by Spore-forming Plant Growth-promoting Rhizobacteria on Cotton

In the past decade, increased attention has been placed on biological control of plant-parasitic nematodes using various fungi and bacteria. The objectives of this study were to evaluate the potential of 662 plant growth-promoting rhizobacteria (PGPR) strains for mortality to Meloidogyne incognita J2 in vitro and for nematode management in greenhouse, microplot, and field trials. Results indicated that the mortality of M. incognita J2 by the PGPR strains ranged from 0 to 100% with an average of 39%. Among the PGPR strains examined, 212 of 662 strains (or 33%) caused significantly greater mortality percent of M. incognita J2 than the untreated control. Bacillus was the major genus initiating a greater mortality percentage when compared with the other genera. In subsequent trials, B. velezensis strain Bve2 reduced M. incognita eggs per gram of cotton root in the greenhouse trials at 45 days after planting (DAP) similarly to the commercial standards Abamectin and Clothianidin plus B. firmus I-1582. Bacillus mojavensis strain Bmo3, B. velezensis strain Bve2, B. subtilis subsp. subtilis strain Bsssu3, and the Mixture 2 (Abamectin + Bve2 + B. altitudinis strain Bal13) suppressed M. incognita eggs per gram of root in the microplot at 45 DAP. Bacillus velezensis strains Bve2 and Bve12 also increased seed-cotton yield in the microplot and field trials. Overall, results indicate that B. velezensis strains Bve2 and Bve12, B. mojavensis strain Bmo3, and Mixture 2 have potential to reduce M. incognita population density and to enhance growth of cotton when applied as in-furrow sprays at planting.

Transcriptome Analysis of Cotton (Gossypium hirsutum L.) Genotypes That Are Susceptible, Resistant, and Hypersensitive to Reniform Nematode (Rotylenchulus reniformis).

Reniform nematode is a semi-endoparasitic nematode species causing significant yield loss in numerous crops, including cotton (Gossypium hirsutum L.). An RNA-sequencing analysis was conducted to measure transcript abundance in reniform nematode susceptible (DP90 & SG747), resistant (BARBREN-713), and hypersensitive (LONREN-1) genotypes of cotton (Gossypium hirsutum L.) with and without reniform nematode infestation. Over 90 million trimmed high quality reads were assembled into 84,711 and 80, 353 transcripts using the G. arboreum and the G. raimondii genomes as references. Many transcripts were significantly differentially expressed between the three different genotypes both prior to and during nematode pathogenesis, including transcripts corresponding to the gene ontology categories of cell wall, hormone metabolism and signaling, redox reactions, secondary metabolism, transcriptional regulation, stress responses, and signaling. Further analysis revealed that a number of these differentially expressed transcripts mapped to the G. raimondii and/or the G. arboreum genomes within 1 megabase of quantitative trait loci that had previously been linked to reniform nematode resistance. Several resistance genes encoding proteins known to be strongly linked to pathogen perception and resistance, including LRR-like and NBS-LRR domain-containing proteins, were among the differentially expressed transcripts mapping near these quantitative trait loci. Further investigation is required to confirm a role for these transcripts in reniform nematode susceptibility, hypersensitivity, and/or resistance. This study presents the first systemic investigation of reniform nematode resistance-associated genes using different genotypes of cotton. The candidate reniform nematode resistance-associated genes identified in this study can serve as the basis for further functional analysis and aid in further development of reniform a nematode resistant cotton germplasm.

Characterization of the two intra-individual sequence variants in the 18S rRNA gene in the plant parasitic nematode, Rotylenchulus reniformis.

The 18S rRNA gene is fundamental to cellular and organismal protein synthesis and because of its stable persistence through generations it is also used in phylogenetic analysis among taxa. Sequence variation in this gene within a single species is rare, but it has been observed in few metazoan organisms. More frequently it has mostly been reported in the non-transcribed spacer region. Here, we have identified two sequence variants within the near full coding region of 18S rRNA gene from a single reniform nematode (RN) Rotylenchulus reniformis labeled as reniform nematode variant 1 (RN_VAR1) and variant 2 (RN_VAR2). All sequences from three of the four isolates had both RN variants in their sequences; however, isolate 13B had only RN variant 2 sequence. Specific variable base sites (96 or 5.5%) were found within the 18S rRNA gene that can clearly distinguish the two 18S rDNA variants of RN, in 11 (25.0%) and 33 (75.0%) of the 44 RN clones, for RN_VAR1 and RN_VAR2, respectively. Neighbor-joining trees show that the RN_VAR1 is very similar to the previously existing R. reniformis sequence in GenBank, while the RN_VAR2 sequence is more divergent. This is the first report of the identification of two major variants of the 18S rRNA gene in the same single RN, and documents the specific base variation between the two variants, and hypothesizes on simultaneous co-existence of these two variants for this gene.

PLANT GROWTH-PROMOTING ACTIVITIES OF BACILLUS SUBTILIS MBI 600 (INTEGRAL ® ) AND ITS COMPATIBILITY WITH COMMONLY USED FUNGICIDES IN RICE SHEATH BLIGHT MANAGEMENT

Sheath blight (ShB) of rice caused by Rhizoctonia solani is an economically important disease, causing significant yield losses. In this study, the growth promoting activities of commercial formulation of a bioagent, Bacillus subtilis MBI 600 (Integral®) and its compatibility with rice fungicides were evaluated. Integral was evaluated for growth promotion in rice on four cultivars (Cocodrie, Catahoula, Neptune, and Trenasse) under in vitro conditions. Treated rice seeds were incubated for 7 days, and the shoot and root lengths were measured. Rice cv. Cocodrie seeds were treated with strain MBI 600 at various concentrations and seeded in pots containing field soil in GH in a randomized complete block design. Germination and seedling lengths were measured at 7 and 15 days after sowing (DAS). The strain MBI 600 was found to produce siderophores. Seed treatment with Integral significantly increased shoot and root lengths at all concentrations in cvs. Cocodrie, Catahoula, and Trenasse under in vitro conditions. The shoot lengths ranged from 39 to 42 mm at a concentration of 2.20 x 109 cfu/ml in all CV’s. At 2.20 x 109 cfu/ml, the root lengths ranged from 47 to 69 mm. The shoot and root lengths of control seedlings were each up to 20 mm. Seed treatment with 2.20 x 108 and 2.20 x 109 cfu/ml significantly increased seedling emergence (81 to 89%) compared to 2.20 x 106 and 2.20 x 107 cfu/ml, and control (61%) under GH conditions. Similarly, seed treatment with 2.20 x 109 cfu/ml of MBI 600 resulted in the highest shoot and root lengths (335 and 166 mm respectively). Integral has good tolerance to hexaconazole, propiconazole, and validamycin; moderate tolerance to tricyclazole; and poor tolerance to benomyl and mancozeb at 1000 ppm. Integral showed compatibility to carbendazim and azoxystrobin up to 400 ppm. Overall, our results suggest that Integral produces siderophores, promoted rice seedling emergence and growth, and is compatible with rice fungicides.

Biological control of Heterodera glycines by spore-forming plant growth-promoting rhizobacteria (PGPR) on soybean

Heterodera glycines, the soybean cyst nematode, is the most economically important plant-parasitic nematode on soybean production in the U.S. The objectives of this study were to evaluate the potential of plant growth-promoting rhizobacteria (PGPR) strains for mortality of H. glycines J2 in vitro and for reducing nematode population density on soybean in greenhouse, microplot, and field trials. The major group causing mortality to H. glycines in vitro was the genus Bacillus that consisted of 92.6% of the total 663 PGPR strains evaluated. The subsequent greenhouse, microplot, and field trials indicated that B. velezensis strain Bve2 consistently reduced H. glycines cyst population density at 60 DAP. Bacillus mojavensis strain Bmo3 suppressed H. glycines cyst and total H. glycines population density under greenhouse conditions. Bacillus safensis strain Bsa27 and Mixture 1 (Bve2 + Bal13) reduced H. glycines cyst population density at 60 DAP in the field trials. Bacillus subtilis subsp. subtilis strains Bsssu2 and Bsssu3, and B. velezensis strain Bve12 increased early soybean growth including plant height and plant biomass in the greenhouse trials. Bacillus altitudinis strain Bal13 increased early plant growth on soybean in the greenhouse and microplot trials. Mixture 2 (Abamectin + Bve2 + Bal13) increased early plant growth in the microplot trials at 60 DAP, and also enhanced soybean yield at harvest in the field trials. These results demonstrated that individual PGPR strains and mixtures can reduce H. glycines population density in the greenhouse, microplot, and field conditions, and increased yield of soybean.

Optimization of In Vitro Techniques for Distinguishing between Live and Dead Second Stage Juveniles of Heterodera glycines and Meloidogyne incognita

Heterodera glycines (Soybean Cyst nematode, or SCN) and Meloidogyne incognita (Root-Knot nematode, or RKN) are two damaging plant-parasitic nematodes on important field crops. Developing a quick method to distinguish between live and dead SCN and RKN second stage juveniles (J2) is vital for high throughput screening of pesticides or biological compounds against SCN and RKN. The in vitro assays were conducted in 96-well plates to determine the optimum chemical stimulus to distinguish between live and dead SCN and RKN J2. Sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), and sodium hydroxide (NaOH) were evaluated for the nematode response to see if these compounds can help distinguish between viable from the dead J2. Results indicated that live SCN J2 responded equally (P ≤ 0.05) to 1 μl Na2CO3 and 10 μl NaHCO3 in 100 μl of water at pH = 10. Live SCN J2 responded by twisting their bodies in a curling shape and increasing rate of movements within 2 minutes of exposure. The twisting activity continued for up to 30 minutes. Live RKN J2 responded by increasing activity with the application of 1 μl NaOH in 100 μl of water at pH = 10 also in the 2 minutes to 30 minutes time frame. Furthermore, in growth chamber tests to confirm the infectivity of live SCN. The live SCN as determined by exposure to 1 μl of Na2CO3 indicated 60.5% of the SCN J2 were alive and of those, 29.5% were infective and entered the soybean roots. The 1 μl of NaOH stimulus revealed that 75.2% RKN J2 were alive and of those, 14.9% were infective and entered soybean roots. These results confirmed that 1 μl of Na2CO3 added to 100 μl suspension of SCN J2 and 1 μl of NaOH added to 100 μl suspension of RKN J2 are the effective stimuli for rapidly distinguishing between live and dead SCN and RKN J2 in vitro. SCN and RKN J2 responded differently to different compounds.

Commercial Potential of Microbial Inoculants for Sheath Blight Management and Yield Enhancement of Rice

Sheath blight of rice is an economically significant disease worldwide. Use of plant growth-promoting rhizobacteria (PGPR), one type of microbial inoculants, for sheath blight management and yield enhancement of rice is gaining popularity in modern agriculture due to increasing concerns with the use of chemical fungicides. Among different microbial inoculants, PGPR are used for their growth-promoting activities and managing sheath blight in rice. However, the efficacy of experimental PGPR strains is typically not consistent under field conditions due to limited knowledge of their formulations, shelf life, delivery systems, compatibility with chemicals and agronomic practices, and the mode of action. In this chapter, a general review on scope and commercial potential of various PGPR for rice sheath blight management and yield enhancement is provided. Efficacy results obtained from tests with Integral®, a current commercial product, which contains the strain Bacillus subtilis MBI600, are presented as an example of the potential for PGPR in management strategies for sheath blight.