Shouan Zhang

Induced Systemic Resistance and Promotion of Plant Growth by Bacillus spp.

ABSTRACT Elicitation of induced systemic resistance (ISR) by plant-associated bacteria was initially demonstrated using Pseudomonas spp. and other gram-negative bacteria. Several reviews have summarized various aspects of the large volume of literature on Pseudomonas spp. as elicitors of ISR. Fewer published accounts of ISR by Bacillus spp. are available, and we review this literature for the first time. Published results are summarized showing that specific strains of the species B. amyloliquefaciens, B. subtilis, B. pasteurii, B. cereus, B. pumilus, B. mycoides, and B. sphaericus elicit significant reductions in the incidence or severity of various diseases on a diversity of hosts. Elicitation of ISR by these strains has been demonstrated in greenhouse or field trials on tomato, bell pepper, muskmelon, watermelon, sugar beet, tobacco, Arabidopsis sp., cucumber, loblolly pine, and two tropical crops (long cayenne pepper and green kuang futsoi). Protection resulting from ISR elicited by Bacillus spp. has been reported against leaf-spotting fungal and bacterial pathogens, systemic viruses, a crown-rotting fungal pathogen, root-knot nematodes, and a stem-blight fungal pathogen as well as damping-off, blue mold, and late blight diseases. Reductions in populations of three insect vectors have also been noted in the field: striped and spotted cucumber beetles that transmit cucurbit wilt disease and the silver leaf whitefly that transmits Tomato mottle virus. In most cases, Bacillus spp. that elicit ISR also elicit plant growth promotion. Studies on mechanisms indicate that elicitation of ISR by Bacillus spp. is associated with ultrastructural changes in plants during pathogen attack and with cytochemical alterations. Investigations into the signal transduction pathways of elicited plants suggest that Bacillus spp. activate some of the same pathways as Pseudomonas spp. and some additional pathways. For example, ISR elicited by several strains of Bacillus spp. is independent of salicylic acid but dependent on jasmonic acid, ethylene, and the regulatory gene NPR1-results that are in agreement with the model for ISR elicited by Pseudomonas spp. However, in other cases, ISR elicited by Bacillus spp. is dependent on salicylic acid and independent of jasmonic acid and NPR1. In addition, while ISR by Pseudomonas spp. does not lead to accumulation of the defense gene PR1 in plants, in some cases, ISR by Bacillus spp. does. Based on the strains and results summarized in this review, two products for commercial agriculture have been developed, one aimed mainly at plant growth promotion for transplanted vegetables and one, which has received registration from the U.S. Environmental Protection Agency, for disease protection on soybean.

Lack of Induced Systemic Resistance in Peanut to Late Leaf Spot Disease by Plant Growth-Promoting Rhizobacteria and Chemical Elicitors

A disease assay was optimized for late leaf spot disease of peanut using Cercosporidium per-sonatum in the greenhouse, and this assay was used in attempts to elicit induced systemic resistance using strains of plant growth-promoting rhizobacteria (PGPR) and chemical elicitors. Nineteen strains of spore-forming bacilli PGPR, including strains of Paenibacillus macerans, Brevibacillus brevis, Bacillus laterosporus, B. subtilis, B. pumilus, B. amyloliquefaciens, B. sphaericus, B. cereus, and B. pasteurii, which previously elicited systemic disease control activity on other crops, were evaluated in greenhouse assays. Seven PGPR strains elicited significant disease reduction in a single experiment; however, none repeated significant protection achieved in the greenhouse assay, while significant protection consistently occurred with the fungicide chlorothalonil (Bravo). In other greenhouse trials, neither stem injections of C. personatum nor foliar sprays of chemicals, including salicylic acid, sodium salicylate, isonicotinic acid, or benzo[1,2,3]thiadiazole-7-carbothioc acid S-methyl ester (Actigard), which elicit systemic acquired resistance on other crops, elicited significant disease protection. In contrast, foliar sprays with DL-β-amino-n-butyric acid (BABA), which is an elicitor of localized acquired resistance, resulted in significantly less late leaf spot disease in one of two tests. Combination treatments of four PGPR strains with BABA in the greenhouse did not significantly protect peanut from late leaf spot. Field trials conducted over two growing seasons indicated that none of the 19 PGPR strains, applied as seed treatments at two concentrations, significantly reduced late leaf spot disease. The same chemical elicitors tested in the greenhouse, including BABA, did not elicit significant disease protection. Some combinations of four PGPR and BABA significantly reduced the disease at one but not at two sample times. Collectively, these results suggest that late leaf spot resistance in peanut is not systemically inducible in the same manner as is resistance to diseases in other crops by PGPR and chemical inducers.

Effect of Application Frequency and Reduced Rates of Acibenzolar-S-Methyl on the Field Efficacy of Induced Resistance Against Bacterial Spot on Tomato

Acibenzolar-S-methyl (ASM), a plant activator known to induce systemic acquired resistance, has demonstrated an ability to manage a number of plant diseases, including bacterial spot on tomato caused by four distinct Xanthomonas spp. The aim of this study was to evaluate application rate and frequency of ASM in order to optimize field efficacy against bacterial spot in Florida, while minimizing its impact on marketable yields. ASM was applied biweekly (once every 2 weeks) as a foliar spray at a constant concentration of 12.9, 64.5, and 129 μM throughout four field experiments during 2007-08. A standard copper program and an untreated control were also included. Overall, biweekly applications of ASM did not significantly reduce disease development or the final disease severity of bacterial spot compared with the copper-mancozeb standard or the untreated control. Only one experiment showed a significant reduction in the final disease severity on plants treated with ASM at 129 μM compared with the untreated control. Three additional field trials conducted during 2009-10 to evaluate the effects of weekly and biweekly applications of ASM at concentrations of 30.3 to 200 μM found that weekly applications provided significantly better disease control than biweekly applications. The tomato yields were not statistically improved with the use of ASM relative to the untreated control and standard copper program. Weekly ASM applications at rates as low as 75 μM (equivalent to 1.58 g a.i./ha in 100 liters of water or 0.21 oz. a.i./acre in 100 gallons of water) to 200 μM (equivalent to 4.20 g a.i./ha in 100 liters of water or 0.56 oz. a.i./acre in 100 gallons of water) were statistically equivalent in managing bacterial spot of tomato without significantly reducing yield compared with the untreated control.

Basil Downy Mildew (Peronospora belbahrii): Discoveries and Challenges Relative to Its Control

Basil (Ocimum spp.) is one of the most economically important and widely grown herbs in the world. Basil downy mildew, caused by Peronospora belbahrii, has become an important disease in sweet basil (O. basilicum) production worldwide in the past decade. Global sweet basil production is at significant risk to basil downy mildew because of the lack of genetic resistance and the ability of the pathogen to be distributed on infested seed. Controlling the disease is challenging and consequently many crops have been lost. In the past few years, plant breeding efforts have been made to identify germplasm that can be used to introduce downy mildew resistance genes into commercial sweet basils while ensuring that resistant plants have the correct phenotype, aroma, and tastes needed for market acceptability. Fungicide efficacy studies have been conducted to evaluate current and newly developed conventional and organic fungicides for its management with limited success. This review explores the current efforts and progress being made in understanding basil downy mildew and its control.

Genetic Diversity of Candidatus Liberibacter asiaticus Based on Two Hypervariable Effector Genes in Thailand

Huanglongbing (HLB), also known as citrus greening, is one of the most destructive diseases of citrus worldwide. HLB is associated with three species of ‘Candidatus Liberibacter’ with ‘Ca. L. asiaticus’ (Las) being the most widely distributed around the world, and the only species detected in Thailand. To understand the genetic diversity of Las bacteria in Thailand, we evaluated two closely-related effector genes, lasA I and lasA II, found within the Las prophages from 239 infected citrus and 55 infected psyllid samples collected from different provinces in Thailand. The results indicated that most of the Las-infected samples collected from Thailand contained at least one prophage sequence with 48.29% containing prophage 1 (FP1), 63.26% containing prophage 2 (FP2), and 19.38% containing both prophages. Interestingly, FP2 was found to be the predominant population in Las-infected citrus samples while Las-infected psyllids contained primarily FP1. The multiple banding patterns that resulted from amplification of lasA I imply extensive variation exists within the full and partial repeat sequence while the single band from lasA II indicates a low amount of variation within the repeat sequence. Phylogenetic analysis of Las-infected samples from 22 provinces in Thailand suggested that the bacterial pathogen may have been introduced to Thailand from China and the Philippines. This is the first report evaluating the genetic variation of a large population of Ca. L. asiaticus infected samples in Thailand using the two effector genes from Las prophage regions.

Evaluation of Microbial Products for Management of Powdery Mildew on Summer Squash and Cantaloupe in Florida

Powdery mildew, caused by Podosphaera xanthii (syn. Sphaerotheca fuliginea auct. p.p.), is a serious and frequently occurring disease of cucurbits worldwide. Efficacy of four microbial products (i.e., Actinovate AG, Companion, BU EXP 1216C, and BU EXP 1216S), which contain microbes as the active ingredient, were evaluated on summer squash and cantaloupe against powdery mildew when applied alone or in alternation with a half-rate of conventional fungicide under greenhouse and field conditions. In greenhouse experiments, the product BU EXP 1216S significantly (P < 0.05) reduced the disease severity by nearly 70% relative to the water control. The level of control achieved was not significantly different from that obtained with Procure 480SC (triflumizole), the half-rate of conventional fungicide treatment, in two of four greenhouse experiments. Compared with the untreated water control, BU EXP 1216C and BU EXP 1216S, when applied alternately with Procure 480SC, consistently promoted plant growth measured by plant height, stem caliper, total fresh weight, and chlorophyll content in the leaves. The degree of increase was 11.6 and 11.3% in plant height, 15.6 and 19.8% in stem caliper, 25 and 40.7% in chlorophyll content, and 164 and 250% in total fresh weight, respectively. Alternating applications of these products with Procure 480SC resulted in significantly less powdery mildew disease than in the water control. In the first field trial on summer squash, all products applied individually or in alternation with Procure 480SC significantly reduced the severity of powdery mildew at the early stage (60 days after planting [DAP]) of disease development. Moreover, these alternating treatments resulted in significantly better control than with Procure 480SC alone at the late assessment stage (88 DAP). The products in alternation with Procure 480SC had a level of disease reduction equivalent to Procure 480SC alone on cantaloupe and significantly reduced disease severity in comparison with the water control. Compared with applying the microbial products alone, alternating applications of these products with Procure 480SC significantly reduced disease severity on cantaloupe and improved the marketable fruit number and weight. The data from our studies suggest that these microbial products could be effectively incorporated into disease management programs. In particular, these microbial products could be integrated into the management of powdery mildew on summer squash and cantaloupe in Florida by alternating their application with low rates of conventional fungicides, potentially reducing the development of fungicide resistance in the pathogen population.

Scirtothrips dorsalis (Thysanoptera: Thripidae): Scanning Electron Micrographs of Key Taxonomic Traits and a Preliminary Morphometric Analysis of the General Morphology of Populations of Different Continents

ABSTRACT The chilli thrips, Scirtothrips dorsalis Hood (Thysanoptera: Thripidae) is an emerging pest of many economically important vegetable and ornamental crops grown in the United States. Accurate identification of this pest is a fundamental requirement in development of effective quarantine and management strategies. Using scanning electron microscopy, high resolution images of important taxonomic traits of this pest were produced, which will aid research, regulatory and extension personnel to identify this pest. High resolution images were obtained for identifying characters of S. dorsalis including tergites with antecostal ridges; head with 3 pairs of ocellar setae, metanotum presenting longitudinal striations with medially located pair of setae; veins of forewing presenting widely spaced setae; segment VIII with complete posteromarginal comb of microtrichia; and sternites lacking discal setae but covered with rows of microtrichia except in the antero-medial region. Further, a preliminary comparison of morphological traits of S. dorsalis populations from different geographical regions was conducted, which can help in understanding the phenotype of this pest. Specimens of S. dorsalis were obtained from 5 distinct geographical regions: New Delhi, India; Shizouka, Japan; Negev, Israel; St. Vincent and Florida in the United States. Fourteen morphological characters of each population of S. dorsalis were measured and compared among the 5 populations. No significant differences were observed between the body lengths of the various S. dorsalis populations, which ranged from 0.85 mm (Negev) to 0.98 mm (Florida). When comparing 12 morphological characters, we found no significant differences among New Delhi, St. Vincent, Negev and Florida populations. However, when S. dorsalis populations of these 4 regions were compared with Shizouka, significant differences were detected for either 2 or 5 morphological characters depending on the population, suggesting the Japan population is more robust i.e., longer and wider mesothorax and metathorax, and wider abdomens. Also, the mean lengths of body size among different populations did not vary directly or inversely with latitude.

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.

Management of Powdery Mildew in Squash by Plant and Alga Extract Biopesticides

Although many fungicides are registered for use to control powdery mildew on cucurbits, management of resistance to fungicides in pathogen populations still remains a major challenge. Two biopesticides Regalia SC and HMO 736 were evaluated in the greenhouse and field for their efficacy against powdery mildew in squash. In greenhouses, Regalia SC alone significantly (P < 0.05) reduced powdery mildew compared to the nontreated control, and was as effective as the chemical standard Procure 480SC (triflumizole). In alternation with Procure 480SC, Regalia SC demonstrated greater or equivalent effects on reducing the disease. HMO 736 alone showed varying levels of disease control, but alternating with Procure 480SC significantly improved control efficacy. In addition, application of Regalia SC or HMO 736 each in alternation with Procure 480SC significantly increased the chlorophyll content in leaves and the total fresh weight of squash plants, when compared with the water control, Regalia SC and HMO 736 alone. In field trials, application of Regalia SC and HMO 736 each alone significantly reduced disease severity in one of two field trials during the early stage of disease development, but not during later stages when disease pressure became high. Both Regalia SC and HMO 736 each applied in alternation with Procure 480SC significantly improved the control efficacy compared to Procure 480SC alone. Results from this study demonstrated that an integrated management program can be developed for powdery mildew in squash by integrating the biopesticides Regalia SC, HMO 736 with the chemical fungicide Procure 480SC.

First Report of Damping-Off on Basella rubra Caused by Rhizoctonia solani Anastomosis Group 4 in Florida

Indian spinach (Basella rubra L.) is a red stem species of Basella that is cultivated worldwide as an ornamental and the aerial parts are also consumed as a vegetable. In May of 2011, symptoms of damping-off were observed on approximately 10% of the plants at the stem base around the soil line of seedlings in a greenhouse in Homestead, FL. Lesions were initially water soaked, grayish to dark brown, irregular in shape, and sunken in appearance on large plants, causing the infected seedlings to collapse and eventually die. Symptomatic stem tissue was surface sterilized with 0.6% sodium hypochlorite, rinsed in sterile distilled water, air dried, and plated on potato dextrose agar (PDA). Plates were incubated at 25°C in darkness for 3 to 5 days. A fungus was isolated in all six isolations from symptomatic tissues on PDA. Fungal colonies on PDA were light gray to brown with abundant growth of mycelia, and the hyphae tended to branch at right angles when examined under a microscope. A septum was always present in the branch of hyphae near the originating point and a slight constriction at the branch was observed. Neither conidia nor conidiophores were found from the cultures on PDA. The characteristics of hyphae, especially the right angle branching of mycelia, indicate close similarity to those of Rhizoctonia solani (2,3). The internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 and sequenced (GenBank Accession No. JN545836). Subsequent database searches by the BLASTN program indicated that the resulting sequence had a 100% identity over 472 bp with the corresponding gene sequence of R. solani anastomosis group (AG) 4 (GenBank Accession No. JF701752.1), a fungal pathogen reported to cause damping-off on many crops. Pathogenicity was confirmed through inoculation of healthy India spinach plants with the hyphae of isolates. Four 4-week-old plants were inoculated with the isolates by placing a 5-mm PDA plug of mycelia at the stem base and covering with a thin layer of the soil. Another four plants treated with sterile PDA served as a control. After inoculation, the plants were covered with plastic bags for 24 h and maintained in a greenhouse with ambient conditions. Four days after inoculation, water-soaked, brown lesions, identical to the symptoms described above, were observed on the stem base of all inoculated plants, whereas no symptoms developed on the control plants. The fungus was isolated from affected stem samples, and the identity was confirmed by microscopic appearance of the hyphae and sequencing the ITS1/ITS4 intergenic spacer region, fulfilling Kochs postulates. This pathogenicity test was conducted twice. R. solani has been reported to cause damping-off of B. rubra in Ghana (1) and Malaysia (4). To our knowledge, this is the first report of damping-off caused by R. solani AG-4 on Indian spinach in Florida and the United States. With the increased interest in producing Asian vegetables for food and ornamental purposes, the occurrence of damping-off on Indian spinach needs to be taken into account when designing programs for disease management in Florida. References: (1) H. A. Dade. XXIX. Bull. Misc. Inform. 6:205, 1940. (2) J. R. Parmeter et al. Phytopathology 57:218, 1967. (3) B. Sneh et al. Identification of Rhizoctonia species. The American Phytopathological Society, St Paul, MN, 1991. (4) T. H. Williams and P. S. W. Liu. Phytopathol. Pap. 19:1, 1976.