scholarly journals Morpho-molecular identification and pathogenicity test on fungal parasites of guava root-knot nematode eggs in Lampung, Indonesia

2020 ◽  
Vol 21 (3) ◽  
Author(s):  
I Gede Swibawa I Gede ◽  
YUYUN FITRIANA ◽  
SOLIKHIN ◽  
RADIX SUHARJO ◽  
F.X. SUSILO ◽  
...  

Abstract. Swibawa IG, Fitriana Y, Solikhin, Suharjo R, Susilo FX, Rani E, Haryani MS, Wardana RA. 2020. Morpho-molecular identification and pathogenicity test on fungal parasites of guava root-knot nematode eggs in Lampung, Indonesia. Biodiversitas 21: 1108-1115. This study aimed to obtain and discover the identity of the species of fungal egg parasites of root-knot nematodes (RKN), which have a high pathogenic ability causing major losses in vegetable crops. The exploration of the fungi was carried out in 2016 and 2018 from Crystal guava plantations in East Lampung, Central Lampung, Tanggamus, and NirAma, a commercial product that has been used for controlling Meloidogyne sp. in Indonesia. Identification was carried out based on morphological characteristics and molecular-based gene sequential analysis of Intergenic Transcribed Spacer (ITS) 1 and ITS 4. A pathogenicity test was carried out in vitro and in a greenhouse using tomato plants as indicator plants. In the in vitro test, observations were made on the percentage of infected RKN eggs. The observations in the greenhouse test were carried out on RKN populations in the soil and roots of tomato plants, root damage (root knots), and damage intensity due to RKN infection. The exploration resulted in five isolates of fungal egg parasites of RKN from the guava plantations in East Lampung (2), Central Lampung (1), Tanggamus (1), and from the isolation results of commercial products (1). The isolates were given codes as B4120X (PT GGP PG1), B3010 (PT GGP PG4), B412G (PT GGP PG 4), B01TG (Tanggamus), and BioP (Commercial products). Based on their morphological characteristics, the isolates were classified into the genus of Paecilomyces. The results of molecular identification showed that the discovered fungi were Purpureocillium lilacinum (Thom.) Luangsa Ard. (Syn. Paecilomyces lilacinus (Thom.) Samson.). Based on the in vitro tests, the five fungal isolates were able to parasitize RKN eggs at 86.4-100%. In the greenhouse test, all isolates significantly suppressed nematode populations in the soil and tomato roots, inhibited the formation of root knots, and produced lower damage intensity compared to controls. Among all the isolates tested, B01TG had the best ability to infect nematode eggs (99.5%), suppressing the formation of root knots, nematode population in the soil and the roots of tomato plants, and the damage intensity compared to other isolates.

2019 ◽  
Vol 32 (1) ◽  
pp. 88-98
Author(s):  
Nuha A. Al-Zehebawi ◽  
Dhia S. Al-Waily ◽  
Labeed A. Al-Saad

The study was designed to examine the effect of manure (poultry wastes) and bio-formulations of Trichoderma harzianum and T. viride separately or with some, to control root-knot disease on radish (Raphanus sativus L.) and chard (Beta vulagaris var.cicla (L.)). The study included the isolation of pathogenic nematode of both plant roots, morphological and molecular identification, examination of the pathogenicity in vitro and in vivo and green house experiments involved application of manure and fungal bio-formulations treatments. The morphological and molecular identification confirmed the identity of root-knot nematode, as Meloidogyne javanica, which was pathogenic to radish and chard. The recorded infection severity was 89 and 95% respectively. The green house experiment results revealed that MThTv treatment was significantly reduced infection severity to 0% for radish and chard in contaminated soil (CS) compared with control positive treatment (55.17 and 40%) respectively. MThTv treatment also showed a highest plant height for Radish in non-nematode-contaminated soil (NCS) and CS treatment (17.85 and 16.50 cm) respectively compared with control positive treatment (5.00 cm), while the highest plant height of Chard was 24.5 cm in MThTv-NCS. The wet weight index in Radish showed a superiority of MThTv and MTh in NC on other treatments (201.75 and 189.5 g.plant-1) respectively followed by MThTv-NCS treatment (184.5 g.plant-1) compared with 19.25 gm.plant-1 in control treatment. In Chard the results showed similar pattern represented by superiority of MThTv-NC treatment (255.25 gm.plant-1) followed by MThTv-NCS (190.75 gm.plant-1) compared with 37.50 gm/plant for positive control.


2019 ◽  
Vol 24 (3) ◽  
pp. 31
Author(s):  
Noor Saad Nahi Al-Adhami ◽  
Alaa Mohsen Al-Araji

The present study aimed to estimate the pathogenesis of Fusarium oxysporum isolates and identify them by molecular technique Polymerase Chain Reaction-Internal Transcript Spacer (PCR-ITS). A total of thirty Fusarium oxysporum isolates were isolated from infected tomato plants and diagnosed depending on 'morphological characteristics. The pathogenicity test was performed for thirty Fusarium oxysporum isolates. So it was procedure steps that Genomic DNA was extracted from seven F. oxysporum isolates according to pathogenicity test by using'' ZR Fungal/Bacterial DNA'' MiniPrep™ kit. Seven F. oxysporum isolates showed high pathogenicity. The concentration and purity of the DNA extracted from the seven F. oxysporum isolates was (150 – 201) ng/µl and (1.4 -1.9) respectively. ITS gene was amplified by using PCR-ITS The sequencing results of amplified product of ITS gene from Fusarium oxysporum isolates indicated that all the seventh sequenced isolates where from one Formae speciales of Fusarium oxysporum as Fusarium oxysporum f. sp. Lycopersici. These results have been devoted to study genetic variability among the species involved in study. Molecular markers are highly important'' to focus on F. oxysporum isolates below'' the species level therefore, the DNA array containing "genus, species'' and forma specialis specific" detector'' for the detection and identification ''of F. oxysporum. Also, Cluster analysis and phylogenetic tree depending on genetic distance revealed the genetic relationship between the thirteen F. oxysporum isolates and confirmed sequencing analysis.   [email protected]


Plant Disease ◽  
2020 ◽  
Author(s):  
Min Li ◽  
Meijiao Hu ◽  
Zhaoyin Gao ◽  
Xiaoyu Hong ◽  
Chao Zhao ◽  
...  

Ipomoea pes-caprae plays an important role in protecting the tropical and subtropical coastal beach of the world. In 2018, a leaf spot was observed on I. pes-caprae in Xisha islands of China, 13.2–25.8% of leaves were infected. The initial symptoms were small (1–3 mm diameter), single, circular, dark gray spots with a light-yellow center on the leaves. The lesions enlarged and were scattered or confluent, distinct and circular, subcircular or irregular, occasionally vein-limited, pale to dark gray-brown, with a narrow dark brown border surrounded by a diffuse yellow margin. Microscopic observations of the spots revealed that caespituli were dark brown and amphigenous, but abundant on the underside of the leaves. Mycelia were internal. Conidiophores were fasciculate, occasionally solitary, pale olivaceous-brown throughout, 0- to 3-septate, 27.9–115.8 (63.4±22.5) µm × 3.2–5.3 (4.3±0.87) µm (n=100). Conidial scars were conspicuously thickened. Conidia were solitary, hyaline, filiform, acicular to obclavate, straight to slightly curved, subacute to obtuse at the apex, truncate at the base, multi-septate, 21.0–125.5 (60.2±20.1) µm × 2.0–5.0 (3.8±0.83) µm (n=100). Single-conidium isolates were obtained from representative colonies grown on potato dextrose agar (PDA) incubated at 25℃ in the dark. The colonies grew slowly and were dense, white to gray and flat with aerial mycelium. Mycelia were initially white, and then became gray. Conidia were borne on the conidiophores directly. The pure isolate HTW-1 was selected for molecular identification and pathogenicity test, which were deposited in Microbiological Culture Collection Center of Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences. The internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1-alpha (tef1) and histone H3 (his3) genes were amplified with ITS1/ITS4, EF-1 / EF-2, and CYLH3F / CYLH3R primers, respectively (Groenewald et al. 2013). The obtained sequences of HTW-1 were all deposited in GenBank with accession numbers MT410467 for ITS, MT418903 for tef1 and MT418904 for his3. The ITS, tef1 and his3 genes all showed 100% similarity for ITS (JX143582), tef1 (JX143340) and his3 (JX142602) with C. cf. citrulina (MUCC 588; MAFF 239409) from I. pes-caprae in Japan. Based on the morphological characteristics and molecular identification, the pathogen was identified as Cercospora cf. citrulina (Groenewald et al. 2013). The pathogenicity test was conducted by spraying conidial suspension (1×104 conidia/mL) on wounded and unwounded leaves for seedling of I. pes-caprae in greenhouse and in sterile vitro condition. The conidial suspension was prepared using conidia from 30-day-old culture grown on PDA at 25℃ in the dark. Leaf surfaces of seedling in greenhouse were wounded by lightly rubbing with a steel sponge and detached leaf surfaces were wounded by sterile needles. the treatments were sprayed with conidial suspensions on wounded and unwounded leaf surfaces. The control was sprayed with sterile water. After eight days, the typical symptoms of spots which were small, single, circular and dark gray appeared on the inoculated wounded leaves, while the inoculated unwounded leaves and the control leaves were symptomless. The pathogen was only re-isolated from the inoculated wounded leaves. The pathogen may be infected by wound. A total of 20 Cercospora and related species was found on Ipomoea spp. (García et al. 1996). Cercospora cf. citrulina has been reported on I. pes-caprae in Japan, although it was unclear if it was a pathogen or saprophyte (Groenewald et al. 2013). To our knowledge, this is the first report of C. cf. citrulina causing leaf spot of I. pes-caprae in China. This disease could threat the cultivation of I. pes-caprae in China.


Nematology ◽  
2001 ◽  
Vol 3 (2) ◽  
pp. 159-164 ◽  
Author(s):  
Yuji Oka

AbstractNematicidal activity of eight essential oil components; trans -anethole, anis alcohol, p-anisaldehyde, benzaldehyde, 4-methoxyphenol, trans-cinnamaldehyde, (R)-(+)-pulegone, 2-furaldehyde, and a non-essential oil component anisole, was tested against the root-knot nematode Meloidogyne javanica in solutions in 200-ml and 3-l pots and in microplots. Among the anisole derivatives, p-anisaldehyde showed the highest nematicidal activity in solutions and in soil. However, trans-cinnamaldehyde, 2-furaldehyde and benzaldehyde showed higher nematicidal activity than p-anisaldehyde in the 3-l pot experiments. EC50 values of trans-cinnamaldehyde for juvenile immobilisation and hatching inhibition in vitro were as low as 15 and 11.3 μl/l, respectively. In the 3-l pot experiments, trans-cinnamaldehyde, 2-furaldehyde, benzaldehyde and carvacrol at a concentration of 100 mg/kg greatly reduced the root galling of tomato, whereas trans-anethole was not effective. In a microplot experiment, soil treatment with trans-cinnamaldehyde (50 ml/m2) reduced the galling index and increased the shoot weight of tomato plants. Although further experiments, such as development of formulations and application methods, are needed, some essential oil components, especially aldehydes, can be developed into lowtoxicity nematicides.


2010 ◽  
Vol 100 (2) ◽  
pp. 199-203 ◽  
Author(s):  
Sergio Echeverrigaray ◽  
Jucimar Zacaria ◽  
Ricardo Beltrão

Nematicidal activity of 22 monoterpenoids were evaluated in vitro and in pot experiments. Twenty of the twenty-two monoterpenoids significantly reduced hatching, and 11 reduced J2 mobility of the root-knot nematode Meloidogyne incognita at a concentration of 250 mg/liter. In general, compounds with hydroxyl and carbonyl groups exhibited higher nematicidal activity than other terpenoids. Borneol, carveol, citral, geraniol, and α-terpineol showed the highest nematicidal activity among the in vitro tested monoterpenoids. These compounds exhibited a dose dependent effect, and drastically reduced eggs hatching and J2 viability at low concentrations. These monoterpenoids, at 100 and 250 mg/kg concentration, diminished root galling of tomato plants in pot experiments. The results suggest that the selected monoterpenoids, and essential oils with high concentration of these compounds, are potential nematicides against Meloidogyne.


2021 ◽  
Vol 12 ◽  
Author(s):  
Rami Kassam ◽  
Jyoti Yadav ◽  
Gautam Chawla ◽  
Aditi Kundu ◽  
Alkesh Hada ◽  
...  

Root-knot nematodes belonging to the genus Meloidogyne are agriculturally important pests, and biocontrol strategies offer safer alternatives for their management. In the present study, two fungal species from Indian soils were identified as Arthrobotrys thaumasia and Tolypocladium cylindrosporum based on morphological characteristics and further confirmed using molecular markers. In vitro evaluation of A. thaumasia against M. incognita and Caenorhabditis elegans showed 82 and 73% parasitism, respectively, whereas T. cylindrosporum gave 65.2 and 57.7% parasitism, respectively. Similarly, culture filtrates of A. thaumasia caused 57.7 and 53.7% mortality of M. incognita and C. elegans, respectively, whereas T. cylindrosporum caused higher mortality of 87.3 and 64%, respectively. Besides, greenhouse evaluation of both fungi against M. incognita infecting tomato significantly reduced nematode disease burden reflecting parasitic success measured as the total number of galls, egg masses, eggs per egg mass, and derived nematode multiplication factor. Application of A. thaumasia and T. cylindrosporum reduced nematode multiplication factor by 80 and 95%, respectively, compared with control. General metabolite profiling of tested fungi using gas chromatography–mass spectrometry and ultra-performance liquid chromatography–quadrupole/time of flight mass spectrometry reported for the first time here showed presence of various volatile and non-volatile compounds with nematicidal activity, viz., trimethyl-heptadiene, methyl-hexadecanol, dodecadienal, decane, terpendole E, dodecane, acetamido-6-anthraquinone, and hexadecanol. Also, other compounds such as undecane, dibutyl-disulfide, octadecenal, paganin, talathermophilin, dactylarin, tolypyridone A, tolypyridone B, pyridoxatin, and destruxin were identified, reported in the literature to possess antibacterial, antifungal, and insecticidal properties. This is the first report of the occurrence of both fungi from India and pioneer demonstration of T. cylindrosporum for root-knot nematode management.


2021 ◽  
Vol 37 (6) ◽  
pp. 596-606
Author(s):  
Pei-Hsuan Wu ◽  
Tung-Tsuan Tsay ◽  
Peichen Chen

Soil-borne diseases are the major problems in mono cropping. A mixture (designated LTM-m) composed of agricultural wastes and a beneficial microorganism Streptomyces saraceticus SS31 was used as soil amendments to evaluate its efficacy for managing Rhizoctonia solani and root knot nematode (Meloidogyne incognita). In vitro antagonistic assays revealed that SS31 spore suspensions and culture broths effectively suppressed the growth of R. solani, reduced nematode egg hatching, and increased juvenile mortality. Assays using two Petri dishes revealed that LTM-m produced volatile compounds to inhibit the growth of R. solani and cause mortality to the root knot nematode eggs and juveniles. Pot and greenhouse tests showed that application of 0.08% LTM-m could achieve a great reduction of both diseases and significantly increase plant fresh weight. Greenhouse trials revealed that application of LTM-m could change soil properties, including soil pH value, electric conductivity, and soil organic matter. Our results indicate that application of LTM-m bio-organic amendments could effectively manage soil-borne pathogens.


2015 ◽  
Vol 33 (2) ◽  
pp. 155-162 ◽  
Author(s):  
Josilda CA Damasceno ◽  
Ana CF Soares ◽  
Fábio N Jesus ◽  
Rosane S Sant'Ana

The effect of sisal liquid residue (fresh and fermented) was evaluated in controlling the root-knot nematode (Meloidogyne javanica) in tomato plants. Bioassays were conducted in vitro with 100 µL of an aqueous suspension containing 300 juveniles (J2) of M. javanica and 1000 µL of sisal liquid residue. The treatments consisted of nematode immersion for 24 and 48 hours in sisal liquid residue, fresh or fermented, diluted in water to the final concentrations of 0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5 and 20%, and nematicide Carbofuran at 350 mg of the active ingredient per liter. Under greenhouse conditions, 4000 juveniles of M. javanica were inoculated on tomato plants grown in pots, and after one week, 100 mL of sisal liquid residue at concentrations of 0, 4, 8, 12, 16 and 20%, were added to soil around the tomato plants. Control treatments received either 100 mL of distilled water or 0.5 g of Carbofuran per pot. Forty days after inoculation, plants were harvested and evaluated for plant growth and root damage. In addition, the selective effect of sisal liquid residue on growth of beneficial soil microorganisms was evaluated. All concentrations of sisal liquid residue presented nematicidal effect, after 48 h of nematode exposure. A mortality rate of 100% was obtained for M. javanica juveniles exposed to liquid residue at a concentration of 20%. Application of increasing concentrations of both sisal liquid residues reduced the number of galls and egg masses per plant and per gram of roots, as well as the final population of M. javanica in soil. Growth of beneficial soil microorganisms was observed in soil amended with sisal fresh liquid residue, for all concentrations tested. The fermented residue caused inhibition of soil beneficial microorganisms. Future studies should be conducted to test the nematicidal effect on tomato plants under field conditions.


Plant Disease ◽  
2021 ◽  
Author(s):  
Mengyi Gu ◽  
Hung Xuan Bui ◽  
Weimin Ye ◽  
Johan Desaeger

Thai basil (Ocimum basilicum var. thyrsiflora) is an important ethnic aromatic herb native to Southeast Asia. According to the Vegetable Production Handbook of Florida 2020-2021, Asian vegetables are currently grown on more than 4,000 ha in Florida, and Thai basil is one of the most commonly grown among these. Meloidogyne spp. cause severe damage to different basil cultivars (Brito et al. 2007). During May-July 2020, plant stunting and galled root symptoms were observed on Thai basil plants sampled from a commercial Asian vegetable farm in Wimauma, Florida (27°44.951' N; 82°16.271’ E); 1,972 root-knot nematode second-stage juveniles (J2s) were extracted from 200 cm3 soil. A pathogenicity test was performed in September 2020 at the University of Florida Gulf Coast Research and Education Center, Wimauma, Florida. Ten of 20, three-week-old nematode-free Thai basil plants were inoculated with 5,000 eggs of field nematode cultures. Two months after inoculation (temperature = 22.8 ± 3.8 °C, relative humidity = 85.6 ± 14.0 %), average gall index (Bridge and Page 1980) = 5.4 ± 1.1 were only observed in inoculated plants, and 69,276 ± 18,904 eggs were extracted from roots using the NaClO method (Hussey and Barker 1973); 5 ± 7 J2s / 200 cc soil were recovered by the modified Baermann funnel technique (Forge and Kimpinski 2007). Nematode reproduction factor (RF) = 13.86 ± 3.78 (Nicol et al. 2010). Morphological measurements (mean, standard deviation and range) of J2s (n=20) included body length = 394.0 ± 22.3 (362.8 - 437.9) µm, body width = 15.7 ± 1.2 (13.6 - 18.3) µm, and stylet length = 12.8 ± 1.1 (10.4-14.5) µm. The perineal pattern of matured female (n=5) was oval-shaped with coarse and smooth striate; the dorsal arch was high and round; no lateral line presented. Morphological characteristics of females and J2s were consistent with those described for M. enterolobii (Yang and Eisenback 1983). DNA was extracted from a single female picked from infected Thai basil root using NaOH digestion method (Hübschen et al. 2004). The D2-D3 expansion segment of 28S rDNA and the COXII region on mitochondrial DNA were amplified by PCR using the primers 28S391a/28S501 and C2F3/1108 (Ye et al. 2020); the species was also confirmed with species-specific primers Me-F/Me-R (Ye et al. 2020). PCR products were sequenced by the Genomic Sciences Laboratory (North Carolina State University, Raleigh, NC, USA) and the results were recorded in the NCBI with GeneBank Accession Nos. MW488150 and MW507374. The sequences showed 100% identity with M. enterolobii in D2/D3 (KP901079, KP411230) and COXII (MN809527, KX214350). M. enterolobii (M. mayaguensis) has been reported on sweet basil in Florida (Brito et al. 2008). To our knowledge, this is the first detection of M. enterolobii on Thai basil in Hillsborough County, Florida. It is not clear to what extent M. enterolobii reduces the yield of Thai basil, but the RF value obtained in the pathogenicity test indicates the crop is certainly a very good host. Limited information is available on the distribution of M. enterolobii in Florida and the US. M. enterolobii is known to break down the root-knot resistance of crops including soybean, sweet potatoes, and tomatoes (Philbrick et al. 2020). This nematode is considered one of the major emerging threats to agriculture in the southeastern US. A multistate research and outreach program (FINDMe program) was initiated in 2019 to study the distribution and management of this nematode in the southeastern US.


Plants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 394
Author(s):  
Reinhard Eder ◽  
Erika Consoli ◽  
Jürgen Krauss ◽  
Paul Dahlin

The devastating root-knot nematode Meloidogyne incognita can cause severe damage to field and greenhouse crops. Due to high economic losses, alternative products are essential to replace banned or strictly regulated nematicides that affect human health and/or the environment. Garlic based products have been previously investigated as environmentally friendly nematicides and their active substances, diallyl polysulfides exist as formulated nematicides on the market. We tested the garlic-based nematicide NEMguard® DE as protective of tomato roots. In vitro evaluation of the lethal concentration (LC) showed strong nematicidal activity with LC50 of 0.8 mg/mL after 96 h and LC90 of 1.5 mg/mL. NEMguard® DE showed protective effect against M. incognita as a single application in small pots and a second application further reduced root galling, significantly. Large greenhouse trials were carried out in two consecutive years to test single and monthly applications of NEMguard® DE. In both years, no controlling effect could be observed on M.incognita. We assume that the silt content of the loamy sandy soil used had an effect on the polysulfides, inhibiting their nematicidal effect. We conclude that further experiments are necessary to investigate the nematicidal potential of NEMguard® DE under different soil compositions or as a different formulation.


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