Seed-borne Fungal Pathogens Research Articles

In-vitro Studies of Plant Extracts against Alternaria brassicae (berk.) Sacc. of Indian Mustard (Brassica juncea (L.) Czern. and coss)

Six plant extract viz., Datura (Daturastramonium), Eucalyptus (Eucalyptus globulous), Karang (Pongamiaglabra),Neem (Azadirachtaindica), Madar (Calotropis gigantean) and Wild sage (Lantana camara) were evaluated in-vitro by poison food technique @ 3, 6, 9 and 12% concentrations against Alternariabrassicaecausing blight of mustard. The results revealed that all the plant extracts inhibited the percent growth inhibition against test fungus as compared to control. However A.indica was found most efficacious with growth inhibition of (38.7%) followed by E. globulus (36.7%). Some extract such as P. glabra and D. stramonium showed moderate inhibition over control (30.9 and 21.6 respectively). Among all plant extract, C. gigantean showed least (15.3%) inhibition over control against A. brassicae. These plant extracts can possibly be used as management of seedborne pathogenic fungi in an eco-friendly way.

Diagnostic methods for detecting fungal pathogens on vegetable seeds

Early diagnosis of seedborne fungal pathogens is particularly important, as often, infected seeds appear symptomless; seed diagnosis can avoid uncontrolled propagation of pathogens through long‐distance exchange of such material. This will prevent economic losses and unnecessary use of fungicides, so reducing costs and the introduction of toxic substances into the environment. Traditional techniques for detection of seedborne fungi are based on incubation and grow‐out methods. Although these are frequently used because of their simplicity of application, they are time‐consuming, require mycological skills, and are sometimes not sensitive enough to low levels of seed infection. Recently, new identification techniques, based on DNA analysis, have been applied and are very efficient due to high sensitivity and specificity. The most common technique is conventional PCR, while other recent techniques include nested PCR, to obviate low levels of target pathogens, multiplex PCR, to detect several pathogens simultaneously, real‐time PCR, to quantify fungi on seeds, and magnetic‐capture hybridization PCR. The main drawbacks of molecular methods are the inability to distinguish between vital and non‐vital inocula, and the difficulty in obtaining quality DNA template, due to PCR inhibitors in seeds. To reduce inhibitory effects, several modified PCR protocols, such as loop‐mediated isothermal amplification, and non‐destructive testing methods have been developed. Loop‐mediated isothermal amplification and next‐generation sequencing have been widely applied in nucleic acid analysis and, given the numerous advantages provided, their application can be substantially extended in the future for detection of fungal pathogens in seeds.

Management of seed borne fungal pathogens of okra collected from seed companies

Effect of mehogoni, mehedi and allamanda extracts were tested to control seed borne fungi of okra seeds collected from 6 companies of notunbazar in Mymensingh district. Prevalence of seed borne fungi was studied by blotter method in the Seed Pathology Center (SPC) and MS Laboratory, Department of Plant Pathology, Bangladesh Agricultural University (BAU), Mymensingh. The highest germination percentage was recorded from ACI seeds (88%), while lowest (70%) in BADC seeds. Six predominant fungal genera were identified. These species were Fusarium oxysporum (5.08%), Aspergillus flavus (4.50%), Aspergillus niger (6.50%), Colletotrichum dematium (4.67%), Rhizopus stolonifer (3.33%) and Penicillium spp. (3.00%). Germination percentage and fungal association varied from company to company. The germination was ranged from 70-95% and infections were recorded 0.80-6.1% in all the treated seeds. Mehogoni extracts at the rate of 1:1 showed best performance in increasing seed germination (96.00%) next to allamanda (70%). Vigour index of okra seeds were increased 19.14% over untreated seeds by the treatment of mehogoni seed extracts at the rate of 1:1. Mehogoniseed extract at the rate of 1:1 seemed to be adoptable at the farmer?s level as an organic management practice.Asian J. Med. Biol. Res. December 2015, 1(3): 628-640

발효식품 추출물과 미생물을 활용한 참깨 종자전염성 병 방제

참깨 ì¢ ìžì „ì—¼ì„± 병해를 ë°©ì œí•˜ê¸° 위하여 다양한 ìœ ìš©ë¯¸ìƒë¬¼ì„ í•¨ìœ í•˜ëŠ” 김치, ê³ ì¶”ìž¥, 된장, 간장, 막걸리를 채집하여 식품 추출물을 ì¡°ì œí•˜ê³ ì¢ ìžì „ì—¼ì„±ë³‘ì›ê· ì— 대한 ì–µì œ 효과를 조사하였다. 추가로 ì‹í’ˆìœ ëž˜ 미생물의 참깨 ì¢ ìžì „ì—¼ì„± 병해 ì–µì œ 효과를 in vitro 및 in vivo 조건하에서 조사하였다. 50개 식품추출물에 처리한 참깨 ì¢ ìžë¥¼ ìƒ¤ë ˆìƒì— 처리하였을 때 20개 식품추출물이 ìœ ë¬˜ì©ìŒì¦ìƒì„ 92% 이상 ê°ì†Œì‹œì¼°ê³ , 7개 식품 추출물 처리는 온실조건에서 지상부 출현율을 58.3-66.8% 향상시키는 것으로 나타났다. 여러 발효식품으로부터 얻은 218 ì¢ ì˜ 발효미생물 중 7ì¢ ì˜ ì¢ ìžì „ì—¼ì„± ë³‘ì›ê· ì— 대하여 높은 í•­ê· í™œì„±ì„ 보였던 29ê· ì£¼ë¥¼ 처리하여 ì¢ ìžì†Œë íš¨ê³¼ë¥¼ ê²€ì • 한 ê²°ê³¼, 20ê· ì£¼ê°€ 병 ì–µì œ 효과가 있는 것으로 나타났다. 20ê· ì£¼ 중 13ê· ì£¼ëŠ” 모썩음 증상을 ìœ ì˜ì„± 있게 감소시켜 ê±´ì „ì£¼ìœ¨ì„ 향상시키는 것으로 나타났다. 16S rDNA region의 염기서열분석을 통해 in vitro 및 in vivo에서 í•­ê· í™œì„± 및 ì¢ ìžì†Œë íš¨ê³¼ë¥¼ 보인 ì‹í’ˆìœ ëž˜ ìœ ìš©ë¯¸ìƒë¬¼ 16ì¢ ì— 대한 ë™ì •ì„ 수행하였다. ê·¸ ê²°ê³¼ 14ê· ì£¼ëŠ” Bacillus 속 ì„¸ê· ìœ¼ë¡œ, 막걸리로부터 분리된 2ê· ì£¼ëŠ” Saccharomyces 속으로 ë™ì •ë˜ì—ˆë‹¤. 발효식품에서 분리된 ì„ ë°œ ê· ì£¼ëŠ” 대부분 B. amyloliquifaciens 인 것으로 나타났다. ì„ ë°œ ê· ì£¼ 중 B. amyloliquefaciens Gcj2-1와 B. amyloliquefaciens Gcj3-1를 ì½”íŒ ì œë¡œ bentonite, kaolin, talc 및 zeolite를 첨가 혼화하여 ì œì œí™”í•˜ì˜€ì„ 때 두 ê· ì£¼ 공히 talc 와 kaolin ì œí˜•ì´ ì–µì œ 효과가 ìš°ìˆ˜í•˜ì˜€ê³ ê·¸ 다음이 zeolite ì œí˜•ì´ì—ˆë‹¤. 그러나 bentonite ì œí˜•ì˜ 경우에는 타 ì œí˜•ì— 비해 ì–µì œ 효과가 낮게 나타났다. ì„ ë°œëœ ë°œíš¨ì‹í’ˆìœ ëž˜ 미생물은 in vitro와 온실조건에서 참깨 ì¢ ìžì „ì—¼ì„±ë³‘ ë°©ì œì— 있어서 효과 ì ì¸ 것으로 확인되었다. 이상의 결과를 근거로 국내산 발효식품 추출물 및 발효식품 ìœ ëž˜ 미생물은 참깨 ì¢ ìžì†Œë ìš© ë°©ì œì œë¡œì„œ ì‚¬ìš©ë ìˆ˜ 있을 것으로 생각된다.

English

Kenaf (Hibiscus cannabinus) is a fibre crop grown in Malaysia as a substitute crop for tobacco. Previous study have recorded that kenaf has been infected by various genera of seed-borne pathogen include Fusarium, Synnematium, Alternaria, Colletotrichum and Botrytis. Seed-borne disease affects and actively attacks seeds and may be harmful. Lasiodiplodia theobromae is a seed-borne fungal pathogen that infects a variety of crop seeds. Studies on the isolation of seed-borne fungi on kenaf seed have revealed that L. theobromae causes black rot disease on kenaf seeds. L. theobromae was successfully isolated from kenaf seeds on an agar plate and a blotter. L. theobromae was isolated frequently from infected seeds and identified based on its cultural and morphological characteristics. The fungus sequence was analysed using molecular technique (ITS-rDNA amplification). A pathogenicity test was used to confirm that L. theobromae caused blackening of the seeds and reduced the germination against a control treatment in potato Dextrose Agar (PDA) medium. To our knowledge, this study is the first to confirm that L. theobromae is the causal agent of black rot on kenaf seed in Malaysia.

Use of silver nanoparticles for managing Gibberella fujikuroi on rice seedlings

It is of great interest for rice producers to develop novel seed disinfection methods with effective, less toxic, and environment-friendly active ingredients. This study evaluated silver nanoparticles (AgNP) as an alternative agent for seed treatment since elemental silver possesses strong antimicrobial properties and a low toxicity to humans and animals. AgNP (∼7.5 nm in diameter) solution was chemically synthesized and evaluated for antifungal activity against Gibberella fujikuroi as representing a seedborne fungal pathogen in rice (Oryza sativa). AgNP reduced viability of G. fujikuroi conidia by 50% when directly exposed to concentrations ranging from 0.015 to 1.5 μg mL−1 for 1 to 20 min. Rice seeds infested with G. fujikuroi and treated with 150 μg mL−1 of AgNP significantly decreased colony-forming units (CFU) of G. fujikuroi on the seed surface when treated for 10 min and up to 24 h. Seed treatment with 150 μg mL−1 AgNP for 12 or 24 h significantly improved seedling emergence and height of G. fujikuroi-infested seeds. Adverse effects on germination rate and seedling growth were not observed with any of the AgNP treatments (≤150 μg mL−1 for up to 48-h exposure) tested in this study. The antifungal effect of AgNP against G. fujikuroi attested in this study suggests that AgNP is a new antifungal ingredient that can be used for managing important seedborne fungal pathogens of rice.

Design of species-specific PCR method for the detection of pathogen Embellisia astragali in standing milk vetch seeds.

Embellisia astragali is an important seedborne pathogenic fungus responsible for yellow stunt and root rot of standing milk vetch (Astragalus adsurgens). The current detection methods that are based on culture and morphological identification are time-consuming, laborious and not always reliable. A PCR-based diagnostic method was developed with the species-specific primer pairs AatpF and AatpR designed from the sequence of the plasma membrane ATPase gene of E. astragali. The specificity of the primers was verified by PCR analysis of DNA from three strains of E. astragali and 19 isolates of nontarget fungi from other genera. A single 135-bp amplicon was detected only from E. astragali isolates, and no cross-reactions were observed with any other tested isolates. The detection limit of this new method was 5 pg of template DNA. The specific primers enabled the detection of E. astragali from the seed of standing milk vetch. This species-specific PCR method provides quick, simple, powerful and reliable detection of E. astragali. PCR-based detection is fast, convenient, precise and sensitive compared to the traditional methods of pathogen detection. This study develops the first PCR method for the detection of Embellisia astragali in standing milk vetch seeds. The species-specific primer set designed from the plasma membrane ATPase gene of E. astragali can detect the pathogen. This assay could be applied in the standing milk vetch seed industry.

In Vitro Studies on Phytochemical Screening of Different Leaf Extracts and Their Antifungal Activity against Seed Borne Pathogen Aspergillus niger

In the present study, screening of aqueous extracts obtained from five medicinally active plants (Melia azedarach, Cassia siamea, Murraya koenigii, Jatropha curcas and Delonix regia) was performed against Aspergillus niger, a causative agent of different destructive diseases of plants. The extracts were also used for the phytochemical screening following Horborne method. The results obtained from this study demonstrated occurrence of alkaloids, tannins, saponins, flavonoids, phenolics, amino acids and terpenes, in the aqueous leaf extract. To obtain an optimum concentration of the extracts, for inhibition of Aspergillus niger, 10%, 15% and 20% concentrations of all the five plants were prepared. The three plants viz., M. azedarach, C. siamea and M. koenigii showed antifungal activity against A. niger in all the concentrations. Inhibition of mycelial growth was lowest at the concentration of 20% of D. regia followed by J. curas. The present protocol showed that the growth of A. niger was inhibited to greater extent at 20% than at lower concentrations of the extracts. It could be suggested that plant extracts could possibly be exploited for the management of seed-borne pathogenic fungi to prevent biodeterioration of seeds in an eco-friendly way.

Draft genome sequence of Phomopsis longicolla isolate MSPL 10-6.

Phomopsis longicolla is the primary cause of Phomopsis seed decay in soybean. This disease severely affects soybean seed quality by reducing seed viability and oil content, altering seed composition, and increasing frequencies of moldy and/or split beans. It is one of the most economically important soybean diseases. Here, we report the de novo assembled draft genome sequence of the P. longicolla isolate MSPL10-6, which was isolated from field-grown soybean seed in Mississippi, USA. This study represents the first reported genome sequence of a seedborne fungal pathogen in the Diaporthe–Phomopsis complex. The P. longicolla genome sequence will enable research into the genetic basis of fungal infection of soybean seed and provide information for the study of soybean–fungal interactions. The genome sequence will also be valuable for molecular genetic marker development, manipulation of pathogenicity-related genes and development of new control strategies for this pathogen.

Efficacy of Seed Washing with Water and Treatment with Garlic Extract and Carbendazim for Reducing Seed-Borne Fungal Pathogens from Rice Variety Kalijira

Seed health of aromatic rice variety Kalijira collected from Mymensingh Sadar Upazilla was studied following standard blotter method. Six different treatments namely water wash, garlic (1 : 1), water wash + garlic (1 : 1), Knowin 50WP (Carbendazim) @ 0.2% and water wash + Knowin-50WP @ 0.2% were used in this experiment. The identified seed-borne fungi were Alternaria padwickii, Aspergillus niger, Aspergillus flavus, Bipolaris oryzae, Curvularia lunata, Fusarium moniliforme, Fusarium oxysporum and Penicillium sp. Highest seed infection (30.67%) was recorded for F. oxysporum and the lowest seed infection (9.33%) was recorded for A. padwickii. Germination of seeds significantly varied in different treatments that ranged from 85.33 to 98.86%. Among the treatments, the highest (98.86%) germination was found by water wash + Knowin-50WP and lowest (85.33%) by control (untreated). After washing of seeds in water, the treated seeds with plant extract or chemical showing better performance than when only treated with plant extract or chemical for controlling seed borne pathogens.DOI: http://dx.doi.org/10.3329/pa.v24i1-2.19098 Progress. Agric. 24(1&2): 45 - 51, 2013

A Non‐thermal Plasma Seed Treatment Method for Management of a Seedborne Fungal Pathogen on Rice Seed

ABSTRACTSeeds contaminated with pathogens are the primary inoculum for plant diseases in many food crops. Conventional treatments for seedborne diseases use hot water, chlorine or fungicide applications. A novel seed treatment method based on non‐thermal plasma generated by an air dielectric barrier discharge (DBD) device was evaluated in this study as an alternative to these conventional treatments. The non‐thermal plasma at atmospheric pressure and room temperature consisted of partially‐ionized gases that are chemically reactive. The antimicrobial activity of non‐thermal plasma was evaluated against an important seedborne pathogen, Gibberella fujikuroi, that causes bakanae disease in rice. Non‐thermal plasma treatments effectively inhibited the growth of G. fujikuroi on nutrient growth medium and reduced the number of fungal colony forming units (CFU) on the rice seed surface by > 92% after 120 s exposure. Effective exposure times to 50% (ET50) and 90% (ET90) control of G. fujikuroi CFU on the seed surface were 9 and 76 s, respectively. Ten minutes of non‐thermal plasma treatment on seeds infested with G. fujikuroi significantly reduced disease development. No adverse effects were detected on the seedling emergence and height when non‐infested seeds were treated with non‐thermal plasma for up to 20 min. The treated seeds germinated and grew similarly compared with non‐treated non‐infested seeds. This study indicates that non‐thermal plasma had antifungal activity and showed potential as an effective disinfection technique to reduce the contamination of seedborne fungal pathogens from seed surfaces without compromising seed health.

Induction of resistance against Cercospora leaf spot in safflower by seed treatment with plant growth-promoting rhizobacteria

Out of seven isolates of rhizobacteria, four strains including GBO-3, INR937a, INR937b and IPC-11 were selected as inducers of systemic resistance against Cercospora carthami and tested individually for biological control of different important seed-borne fungal pathogens. The level of control achieved by each rhizobacterium varied with the pathosystem studied. The isolates GBO-3, INR937a, INR937b and IPC-11 exhibited reduced disease intensity in terms of average number of leaf lesions as compared to untreated control in protection experiments against C. carthami. Micromobilised with rhizobacterium GBO-3, INR937a, INR937b and IPC-11 safflower seeds were grown under greenhouse and challenge inoculated with C. carthami were selected for estimation of lipoxygenase, polyphenylalanine ammonia-lyase, peroxidase, polyphenol oxidase and β-1,3 glucanase activities were estimated spectrophotometrically. Increase in all enzymes was detected in foliar extracts from plants at different stages after challenge inoculation.

Seed disinfection effect of atmospheric pressure plasma and low pressure plasma on Rhizoctonia solani.

Gas plasma generated and applied under two different systems, atmospheric pressure plasma and low pressure plasma, was used to investigate the inactivation efficacy on the seedborne pathogenic fungus, Rhizoctonia solani, which had been artificially introduced to brassicaceous seeds. Treatment with atmospheric plasma for 10 min markedly reduced the R. solani survival rate from 100% to 3% but delayed seed germination. The low pressure plasma treatment reduced the fungal survival rate from 83% to 1.7% after 10 min and the inactivation effect was dependent on the treatment time. The seed germination rate after treatment with the low pressure plasma was not significantly different from that of untreated seeds. The air temperature around the seeds in the low pressure system was lower than that of the atmospheric system. These results suggested that gas plasma treatment under low pressure could be effective in disinfecting the seeds without damaging them.

Study of seed-borne fungal pathogens of Kataribhog aromatic rice and comparison of field intensity with laboratory counts

Study of seed-borne fungal pathogens of Kataribhog aromatic rice and comparison of field intensity with laboratory counts

Seed treatments to control seedborne fungal pathogens of vegetable crops

Vegetable crops are frequently infected by fungal pathogens, which can include seedborne fungi. In such cases, the pathogen is already present within or on the seed surface, and can thus cause seed rot and seedling damping-off. Treatment of vegetable seeds has been shown to prevent plant disease epidemics caused by seedborne fungal pathogens. Furthermore, seed treatments can be useful in reducing the amounts of pesticides required to manage a disease, because effective seed treatments can eliminate the need for foliar application of fungicides later in the season. Although the application of fungicides is almost always effective, their non-target environmental impact and the development of pathogen resistance have led to the search for alternative methods, especially in the past few years. Physical treatments that have already been used in the past and treatments with biopesticides, such as plant extracts, natural compounds and biocontrol agents, have proved to be effective in controlling seedborne pathogens. These have been applied alone or in combination, and they are widely used owing to their broad spectrum in terms of disease control and production yield. In this review, the effectiveness of different seed treatments against the main seedborne pathogens of some important vegetable crops is critically discussed.

Percolation-Based Risk Index for Pathogen Invasion: Application to Soilborne Disease in Propagation Systems

Propagation systems for seedling growth play a major role in agriculture, and in notable cases (such as organic systems), are under constant threat from soil and seedborne fungal plant pathogens such as Rhizoctonia solani or Pythium spp. Yet, to date little is known that links the risk of disease invasion to the host density, which is an agronomic characteristic that can be readily controlled. We introduce here, for the first time in an agronomic system, a percolation framework to analyze the link. We set up an experiment to study the spread of the ubiquitous fungus R. solani in replicated propagation systems with different planting densities, and fit a percolation-based epidemiological model to the data using Bayesian inference methods. The estimated probability of pathogen transmission between infected and susceptible plants is used to calculate the risk of invasion. By comparing the transmission probability and the risk values obtained for different planting densities, we are able to give evidence of a nonlinear relationship between disease invasion and the inter-plant spacing, hence to demonstrate the existence of a spatial threshold for epidemic invasion. The implications and potential use of our methods for the evaluation of disease control strategies are discussed.

Seed borne fungal pathogens associated with common bean (Phaseolus vulgaris L.) seeds and their impact on germination

Seed borne fungal pathogens associated with common bean (Phaseolus vulgaris L.) seeds and their impact on germination

English

Management and transmission of four seed-borne pathogenic fungi namely,Colletotrichum trifolii,  Rhizoctonia solani, Fusarium equiseti and F. incarnatum were investigated on alfalfa plants. The pathogenicity test on alfalf plant showed that R. solaniand C. trifolii caused  high percentages of rotted seeds and seedlings mortality (26.45, 26.1% and 31.6,  21.1% , respectively). However, no significant differences were observed between the treatments with F. equiseti and F. incarnatum when compared with the control treatment. Transmission of the pathogenic fungi from seed to mature plant of alfalfa was investigated in this study. Results indicate that the recovery percentages of the tested pathogens gradually decreased from root apex up to the first internodes below the shoot tip, but did not reach to the shoot apex. In order to control these fungi, different concentrations of sodium metabisulphite (SM), sodium salicylate (SS) and hydroquinone (HQ) were tested in vitro. Treatment with SM at 10 mM completely inhibited the growth of all isolated fungi. Under greenhouse conditions, soaking alfalfa seeds in a water solution of hydroquinone at 12mM showed to be the most effective treatment in reducing seed rot and seedling mortality percentages and increasing seedling survival percentages. Application of the tested treatments presented significant increases in growth parameters, photosynthetic pigments (chlorophyll a and b and carotenoids) in leaves and total phenol in alfalfa plants. This study therefore recomended the use of HQ and SM as potential and promising antifungal agents in  the protection of alfalfa plants against the tested seed-borne fungi.   Key words: Pathogenicity, Colletotrichum trifolii, Rhizoctonia solani, Fusarium equiseti, F. incarnatum, transmission, hydroquinone, sodium metabisulphite and sodium salicylate.

Efficacy of essential oils as jute seed protectant

Essential oils obtained from the leaves of Eucalyptus citriodora, E. camaldulensis, and whole plants of Ocimum gratissimum and O. americanum were tested for their efficiency to protect seed of jute (var.D-154) from seed-borne fungal pathogens. A total of 9 fungi were detected. Among the 9 deteced seed borne pathogenic fungi, the highest percentage of fungal occurrence was recorded with Macrophomina phaseolina (37.71%). This was followed by Botryodiplodia theobromae (30.10%) and Colletotrichum corchori (19.72%). The jute seeds dressed with the oil of Eucalyptus citriodora and Ocimum gratissimum exibited complete elimination of seed fungi, increased seed germination and reduced seedling mortality as compared to that of E. camaldulensis and O. americanum.DOI: http://dx.doi.org/10.3329/cujbs.v5i1.13364 The Chittagong Univ. J. B. Sci.,Vol. 5(1 &2):01- 07, 2010

Specific PCR-based detection of Alternaria helianthi: the cause of blight and leaf spot in sunflower

Alternaria helianthi is an important seed-borne pathogenic fungus responsible for blight disease in sunflower. The current detection methods, which are based on culture and morphological identification, are time-consuming, laborious and are not always reliable. A PCR-based diagnostic method was developed with species-specific primers designed based on the sequence data of a region consisting of the 5.8S RNA gene and internal transcribed spacers-ITS 1 and ITS 2 of nuclear ribosomal RNA gene (rDNA) repeats of A. helianthi. The specificity of the primer pairs AhN1F and AhN1R designed was verified by PCR analysis of DNA from 18 Alternaria helianthi strains isolated from India, 14 non-target Alternaria spp. and 11 fungal isolates of other genera. A single amplification product of 357-bp was detected from DNA of A. helianthi isolates. No cross-reaction was observed with any of the other isolates tested. The detection limit of the PCR method was of 10pg from template DNA. The primers could also detect the pathogen in infected sunflower seed. This species-specific PCR method provides a quick, simple, powerful and reliable alternative to conventional methods in the detection and identification of A. helianthi. This is the first report of an A. helianthi-specific primer set.