Suppression of maize root diseases caused by Macrophomina phaseolina, Fusarium moniliforme and Fusarium graminearum by plant growth promoting rhizobacteria.

A plant growth-promoting isolate of a fluorescent Pseudomonas sp. EM85 and two bacilli isolates MR-11(2) and MRF, isolated from maize rhizosphere, were found strongly antagonistic to Fusarium moniliforme, Fusarium graminearum and Macrophomina phaseolina, causal agents of foot rots and wilting, collar rots/stalk rots and root rots and wilting, and charcoal rots of maize, respectively. Pseudomonas sp. EM85 produced antifungal antibiotics (Afa+), siderophore (Sid+), HCN (HCN+) and fluorescent pigments (Flu+) besides exhibiting plant growth promoting traits like nitrogen fixation, phosphate solubilization, and production of organic acids and IAA. While MR-11(2) produced siderophore (Sid+), antibiotics (Afa+) and antifungal volatiles (Afv+), MRF exhibited the production of antifungal antibiotics (Afa+) and siderophores (Sid+). Bacillus spp. MRF was also found to produce organic acids and IAA, solubilized tri-calcium phosphate and fixed nitrogen from the atmosphere. All three isolates suppressed the diseases caused by Fusarium moniliforme, Fusarium graminearum and Macrophomina phaseolina in vitro. A Tn5:: lacZ induced isogenic mutant of the fluorescent Pseudomonas EM85, M23, along with the two bacilli were evaluated for in situ disease suppression of maize. Results indicated that combined application of the two bacilli significantly (P = 0.05) reduced the Macrophomina-induced charcoal rots of maize by 56.04%. Treatments with the MRF isolate of Bacillus spp. and Tn5:: lacZ mutant (M23) of fluorescent Pseudomonas sp. EM85 significantly reduced collar rots, root and foot rots, and wilting of maize caused by Fusarium moniliforme and F. graminearum (P = 0.05) compared to all other treatments. All these isolates were found very efficient in colonizing the rhizotic zones of maize after inoculation. Evaluation of the population dynamics of the fluorescent Pseudomonas sp. EM85 using the Tn5:: lacZ marker and of the Bacillus spp. MRF and MR-11(2) using an antibiotic resistance marker revealed that all the three isolates could proliferate successfully in the rhizosphere, rhizoplane and endorhizosphere of maize, both at 30 and 60 days after seeding. Four antifungal compounds from fluorescent Pseudomonas sp. EM85, one from Bacillus sp. MR-11(2) and three from Bacillus sp. MRF were isolated, purified and tested in vitro and in thin layer chromatography bioassays. All these compounds inhibited R. solani, M. phaseolina, F. moniliforme, F. graminearum and F. solani strongly. Results indicated that antifungal antibiotics and/or fluorescent pigment of fluorescent Pseudomonas sp. EM85, and antifungal antibiotics of the bacilli along with the successful colonization of all the isolates might be involved in the biological suppression of the maize root diseases.     

Occurrence of Charcoal Rot Caused by Macrophomina phaseolina,an Emerging Disease of Adzuki Bean in China

Adzuki bean (Vigna angularis) is an important legume crop in China. Soil‐borne charcoal rot caused by Macrophomina phaseolina (Tassi) Goid is an important and devastating disease of many crops including legumes. During late August and early September, 2014, symptoms similar to charcoal rot were observed on adzuki bean plants in Yulin City of Shanxi Province, and Fangshan County of Beijing, China. This study was conducted to determine the causal agent of the emerging disease on adzuki bean. Four fungal isolates were obtained and identified as M. phaseolina based on morphological and molecular characteristics, including species‐specific primer detection and sequences of internal transcribed spacer (ITS) of nuclear ribosomal DNA. The resulting sequences showed 99% identity with more than 60 M. phaseolina strains from diverse hosts. The virulence on adzuki bean was verified using pathogenicity tests, producing symptoms similar to those observed in the fields. To our knowledge, this is the first report of M. phaseolina causing charcoal rot on adzuki bean.     

Effects of Nitrogen, Plant Density, Moisture Stress and Artificial Inoculation with Macrophomina phaseolina on Charcoal Rot Incidence in Grain Sorghum

The occurrence of pythiaceous fungi in pot plant cultures grown in ebb and flow bench systems was investigated monthly from May to December, Phytophthora, Pythium and Saprolegnia (in all 351) were isolated from water samples and identified. Nearly all the isolates of Pythium produced zoospores in water. A pathogenicity test involving 15 isolates of Pythium“group P”, and 7 of Pythium“group F” showed that 73 % were pathogenic on cucumber, 66 % on Gerbera, 59 % on lettuce, 50 % on tomato, and 32 % on cress. Control of Pythium and Phytophthora is important in order to improve the health of plants grown in ebb and flow systems.     

Physical Factors that Influence the Growth and Spread of Charcoal Rot Pathogen (Macrophomina phaseolina) Infecting Maize

The rabi maize, that is being popularised in the eastern parts of the country may suffer from charcoal rot disease [Rhizoctonia bataticola Taub. Butl. (Macrophomina phaseolina (Tassi) Goid.)] if the pathogen gets timely entry into the host, as has been revealed in the present study. In vitro studies show that the spread of the pathogen within the maize stalk is influenced by high temperature, the optimum being 38°C. The role of RH on the incidence of this disease was, however, not well defined. These observations hold true as well for the data recorded in the field.     

Molecular characterization of Macrophomina phaseolina and Fusarium species by a single primer RAPD technique

Charcoal root rot and wilt, are two economically important diseases of many crop plants in North and South America, Asia and Africa and some parts of Europe. Genetic variation in 43 isolates of Macrophomina phaseolina and 22 isolates of Fusarium species, collected from geographically distinct regions over a range of hosts, was studied using random amplified polymorphic DNA (RAPD) markers. Initially, 210 arbitrary nucleotide (10-mer) primers were tested for amplification of genomic DNA of one M. phaseolina isolate, 70 primers amplified the genomic DNA of M. phaseolina. One primer OPA-13 (5'-CAGCACCCAC-3') produced fingerprint profiles, which clearly distinguished between the different isolates of M. phaseolina. UPGMA analysis classified these isolates into five major groups. By primer OPA-13, 22 isolates of pathogenic and non-pathogenic Fusarium species of different formae-speciales and races, were also distinguished from M. phaseolina. This marker is useful for distinguishing between these two important plant pathogens irrespective of hosts, virulence spectrum and races. This is the first report of reliable diagnosis of two soilborne pathogens (root/collar rot and wilt causing pathogens) at the level of isolates, formae-speciales and races by a single primer RAPD procedure with uniform PCR conditions.     

Seed Treatment with Live or Dead Fusarium verticillioides Equivalently Reduces the Severity of Subsequent Stalk Rot

Fusarium verticillioides is a widely distributed fungus that can associate with maize as a deleterious pathogen and an advantageous endophyte. Here, we show that seed treatment with live F. verticillioides enhances maize resistance to secondary stalk rot infection and further demonstrate that dead F. verticillioides is sufficient to equivalently reduce F. verticillioides biomass. Seed treatment with live or dead F. verticillioides primes maize plants, and upon subsequent stalk infection, terpenoid phytoalexins accumulate faster than control‐treated plants. Seed treatment did not constitutively activate plant defences nor did it impact plant growth. These results suggest that seed treatment with dead F. verticillioides can be used as a ‘vaccination’ method to decrease the severity of stalk rot and potentially pathogen infection throughout the plant.     

Species-specific PCR Assays for the Fungal Pathogens Fusarium moniliforme and Fusarium subglutinans and their Application to Diagnose Maize Ear Rot Disease

Fusarium moniliforme Sheldon (syn. F. verticillioides (Sacc.) Nirenberg) and F. subglutinans (Wollenweber & Reinking) Nelson Toussoun & Marasas comb. nov., two anamorphs of the so-called‘Gibberella fujikuroi species complex', are important maize pathogens. Together with F. proliferatum, F. culmorum, and F. graminearum (teleomorph: Gibberella zeae) they are involved in the stalk rot and ear rot disease of maize. All species produce secondary metabolites (mycotoxins) which are a potential health hazard for humans and animals that consume maize and maize products frequently. In this study the development of polymerase chain reaction (PCR) assays for an easy and sensitive identification of G. fujikuroi anamorphs in maize kernels are described. The primer pairs are based on sequences of randomly amplified polymorphic DNA (RAPD) fragments and are specific for F. moniliforme and F. subglutinans respectively. The PCR assays are independent of the high phenotypic variability of traits which may complicate classification by morphological characters. They detect approximately 100 to 200 fungal genomes in the presence of an excess of maize DNA. For the analysis of infected maize kernels a rapid and easy DNA extraction was used which does not introduce inhibitory substances into the PCR. Hence the assays enable an early identification and detection of the two pathogens in host tissue by plant breeders and plant health inspection services. The assays were successfully applied to identify field isolates from Poland and to detect the pathogens in maize ears of various hybrids in Germany.     

Incidence of Stenocarpella and Fusarium Cob Rots in Monoculture Maize under Different Tillage Systems

The incidence of maize cob rot caused by Stenocarpella maydis, S. macrospora, Fusarium moniliforme, F. subglutinans and F. graminearum was determined over two seasons under different tillage systems at various localities. Tillage had no effect on Fusarium spp. cob rots. S. macrospora occurred only at one locality, viz. Cedara, and no tillage effect was observed. Ploughing reduced the incidence of S. maydis cob rot at localities which had high incidences of disease. The relationship between severity of S. maydis cob rot and surface stubble mass was linear.     

Interaction of Meloidogyne javanica and Macrophomina phaseoli in Kenaf Root Rot

Incidence and severity of root-rot caused by the fungus Macrophomina phaseoli was increased in screenhouse-grown kenaf (Hibiscus cannabinus L.) seedlings simultaneously infected by the nematode Meloidogyne javanica. In seedlings inoculated at 5, 10 and 15 days of age, root rot lesions increased 70.3, 44.1 and 21.8%, and nematode penetration increased 49.0, 36.7, and 12.3% when both fungus and nematode were present.     

Evaluation of Durum Wheat Genotypes for Resistance against Root Rot Disease Caused by Moroccan Fusarium culmorum Isolates

Fusarium culmorum is one of the most important causal agents of root rot of wheat. In this study, 10 F. culmorum isolates were collected from farms located in five agro-ecological regions of Morocco. These were used to challenge 20 durum wheat genotypes via artificial inoculation of plant roots under controlled conditions. The isolate virulence was determined by three traits (roots browning index, stem browning index, and severity of root rot). An alpha-lattice design with three replicates was used, and the resulting ANOVA revealed a significant (P < 0.01) effect of isolate (I), genotype (G), and G × I interaction. A total of four response types were observed (R, MR, MS, and S) revealing that different genes in both the pathogen and the host were activated in 53% of interactions. Most genotypes were susceptible to eight or more isolates, while the Moroccan cultivar Marouan was reported resistant to three isolates and moderately resistant to three others. Similarly, the Australian breeding line SSD1479-117 was reported resistant to two isolates and moderately resistant to four others. The ICARDA elites Icaverve, Berghisyr, Berghisyr2, Amina, and Icaverve2 were identified as moderately resistant. Principal component analysis based on the genotypes responses defined two major clusters and two sub-clusters for the 10 F. culmorum isolates. Isolate Fc9 collected in Khemis Zemamra was the most virulent while isolate Fc3 collected in Haj-Kaddour was the least virulent. This work provides initial results for the discovery of differential reactions between the durum lines and isolates and the identification of novel sources of resistance.     

Effects of Temperature and Moisture on Development of Fusarium graminearum Perithecia in Maize Stalk Residues

Fusarium graminearum is the predominant component of the Fusarium head blight complex of wheat. F. graminearum ascospores, which initiate head infection, mature in perithecia on crop residues and become airborne. The effects of temperature (T) and moisture on perithecium production and maturation and on ascospore production on maize stalk residues were determined. In the laboratory, perithecia were produced at temperatures between 5 and 30°C (the optimum was 21.7°C) but matured only at 20 and 25°C. Perithecia were produced when relative humidity (RH) was ≥75% but matured only when RH was ≥85%; perithecium production and maturation increased with RH. Equations describing perithecium production and maturation over time as a function of T and RH (R² > 0.96) were developed. Maize stalks were also placed outdoors on three substrates: a grass lawn exposed to rain; a constantly wet, spongelike foam exposed to rain; and a grass lawn protected from rain. No perithecia were produced on stalks protected from rain. Perithecium production and maturation were significantly higher on the constantly wet foam than on the intermittently wet lawn (both exposed to rain). Ascospore numbers but not their dispersal patterns were also affected by the substrate.     

Phytophthora Crown and Root Rot of Apricot Caused by Phytophthora palmivora in Turkey

Forty‐nine Phytophthora isolates were obtained from roots and crown of apricot trees with symptoms of decline grown in commercial orchards in Malatya, Elaz?? and Diyarbak?r provinces, Turkey, in 2011 and 2013. All of the recovered isolates were identified as Phytophthora palmivora on the basis of morphological characteristics. Blast analysis of ITS region sequences of rDNA of 5 isolates revealed 100% identity with a reference isolates of P. palmivora from GenBank. Isolates of P. palmivora were pathogenic on 12‐month‐old wild apricot rootstock ‘Zerdali’ plants that were wound inoculated on the roots and on the crown. This study demonstrated that P. palmivora is the cause of the crown and root rot found on apricot in Turkey. To our knowledge, this is the first report of P. palmivora on this host plant.     

Effects of Preconditioning Through Mycorrhizal Inoculation on the Control of Melon Root Rot and Vine Decline Caused by Monosporascus cannonballus

The use of inoculum of arbuscular mycorrhizal fungi (AMF) in nursery represents a promising field in horticulture because of its known benefits in terms of plant growth and bioprotection. The present work was undertaken to determine the effect of mycorrhizal inoculation with Rhizophagus irregularis in a nursery medium on the containment of melon root rot and vine decline (MRRVD) caused by the soil‐borne pathogen Monosporascus cannonballus. The percentage of mycorrhization, biomass and yield following mycorrhizal inoculation were also evaluated. Biocontrol activity was assessed in greenhouse pot experiments upon artificial inoculation of M. cannonballus and in a two‐season field experiment under production conditions in an unheated greenhouse with a history of MRRVD. On the basis of the mycorrhization parameters, the interaction appeared to be established within 30 days after inoculation. The total shoot growth in the mycorrhized plants was significantly higher when compared to the control, while the root growth was unaffected. Upon artificial inoculation of M. cannonballus, mycorrhization provided complete protection against the pathogen. Greenhouse experiments under production conditions during spring cropping season showed that pretransplanting inoculation with R. irregularis significantly decreased the severity of the disease. Also, the average fruit weight of mycorrhized plants was significantly higher than the untreated control. Nevertheless, in summer crop, the bioprotection activity of AMF failed. Present results indicate that the use of AMF in a nursery setting can contribute to the prevention of the onset of this problematic soil‐borne disease within a sustainable and integrated soil‐borne disease management.     

Effect of different temperature and culture media on the growth of Macrophomina phaseolina

The charcoal root disease caused by Macrophomina phaseolina (Tassi) Goidanich may cause considerable damages in hot as well as in dry seasons. The effect of temperature and culture media were studied on the growing patterns of 35 M. phaseolina isolates, collected from different districts of Hungary. The isolates were grown at 10, 15, 20, 25, 30, 35 and 40 degrees C temperatures respectively, and additionally at 25 degrees C on potato-dextrose-, malt-extract-, Czapek-Dox-, Sabouraud-glucose-, maize-flour- and watery agar media, using 90 mm Petri-dishes, 4 repetitions in each case. For all the isolates the most favourable temperature regime was 25 to 35 degrees C and the most advantageous media was the malt-extract-, Sabouraud-glucose- and potato-dextrose-agar media. At these conditions (temperatures and culture media) mycelia growth and the diameter of microsclerotial colonies reached the 90 mm at the 5th day. Mycelia growth of the pathogen was very low at 10, 15 and 40 degrees C, and did not form microsclerotia. On watery agar microsclerotial colony seldom developed, it needed 14 days, and no continuous mycelia developed even in a 8th months culture. Diameter of microsclerotia measured on different culture media varied between 39-308 microm.     

Rapid Diagnosis of Soya Bean Root Rot Caused by Fusarium culmorum Using a Loop‐Mediated Isothermal Amplification Assay

We report a rapid diagnosis of soya bean (Glycine max L.) root rot caused by Fusarium culmorum, using a loop‐mediated isothermal amplification (LAMP) assay. We used the CYP51C gene sequence to design LAMP assay primers specific for F. culmorum. The LAMP assay amplified the target gene efficiently in 60 min at 63°C. The sensitivity of the assay was 100 pg/μl of genomic DNA. Among the tested soya bean pathogens, a positive colour (sky blue) was only observed in the presence of F. culmorum with the addition of hydroxynaphthol blue (HNB) dye prior to amplification, whereas other species isolates showed no colour change. Suspected diseased soya bean samples collected in the field from Jiangsu, Shandong and Anhui provinces and Beijing were diagnosed successfully using the LAMP assay reported here. This study provides a new and readily available method for rapid diagnosis of soya bean root rot caused by F. culmorum.