Rice nematode problems in Nigeria: Their occurrence,distribution and pathogenesis

Abstract

In a survey of plant parasitic nematodes associated with or affecting rice throughout Nigeria, some important nematode pests, especially the white tip disease nematode, Aphelenchoides besseyi and the rice root nematodes Hirschmanniella spinicaudata and H. oryzae were identified from seed, soil and root samples from swamp rice fields respectively. The sugarcane cyst nematode, Heterodera sacchari occurred in swamp rice fields only around the major sugarcane estates in Nigeria. The root‐knot Meloidogyne incognita and the root lesion nematode, Pratylenchus brachyurus were also encountered in upland (rainfed) rice fields. The white tip disease nematode, A. besseyi occurred at low levels in soils and rice seeds throughout the country. High population levels of H. spinicaudata and H. oryzae were encountered especially in areas where monoculture of rice is practised. General chlorosis, poor tillering and significantly reduced yield have been observed due to H. spinicaudata. Rice plants attacked by H. sacchari also showed intense chlorosis, delayed and reduced tillering and reduced grain yield. The roots of attacked plants were twiggy, very necrotic and blackened. The root‐knot M. incognita and the root lesion nematodes P. brachyurus have both been observed to reduce rice yields. Rice cultivars screened for reactions to the nematodes showed varying degrees of susceptibilities. Some varieties were however resistant to the root‐knot nematode, M. incognita.     

河南省禾谷类作物孢囊线虫的发生分布与种类鉴定

为探究河南省主要禾谷类作物孢囊线虫的发生分布,明确孢囊线虫对不同作物的危害情况,于2017—2020年对河南省18个市50个县（区）的小麦、玉米和水稻作物的孢囊线虫种类和发生分布进行随机取样调查,采用形态学特征、分子生物学鉴定和rDNA-ITS序列进化树分析技术鉴定不同作物孢囊线虫的种类,并根据土壤中孢囊基数和单孢囊卵数明确不同作物孢囊线虫的发病严重度。结果显示,共采集土壤样品308份,其中224份样品检测到孢囊,孢囊检出率为72.7%。小麦孢囊线虫发生分布范围覆盖40个县（区）,其中15个县（区）为禾谷孢囊线虫Heterodera avenae侵染,23个县（区）为菲利普孢囊线虫H. filipjevi侵染,南阳市西峡县西坪镇和开封市尉氏县张市镇为禾谷孢囊线虫和菲利普孢囊线虫混合侵染发生区;玉米孢囊线虫H. zeae在濮阳市清丰县韩村镇、许昌市长葛市董村镇和禹州市范坡镇检测点首次被发现;旱稻孢囊线虫H. elachista在信阳市潢川县魏岗乡、来龙乡和新乡市获嘉县亢村镇检测点首次被发现。孢囊线虫发病严重度数据表明,小麦田平均孢囊含量高达17.3个/100 mL;玉米田平均孢囊量为11.0个/100 mL;水稻田平均孢囊量为4.4个/100 mL。表明河南省孢囊线虫高发地块主要集中在豫北、豫东和豫中平原区。     

近年水稻主要线虫病害的研究进展

目前,以拟禾谷根结线虫Meloidogyne graminicola、水稻干尖线虫Aphelenchoide besseyi、水稻潜根线虫Hirschmanniella oryzae、水稻茎线虫Ditylenchus angustus和旱稻孢囊线虫Heterodera elachista为主的水稻寄生线虫侵染水稻以及其它禾本科作物引致的线虫病害,已造成了严重的经济损失。近年来,对拟禾谷根结线虫的致病机制和寄主的防御机制等方面的研究已取得了显著进展,但对其它水稻寄生线虫与寄主互作机制的研究还存在较大空白。水稻寄生线虫的防治多依赖化学农药,抗性品种和生物防治等措施的实施存在一定的局限性。本文对上述5种主要水稻寄生线虫病的发生为害、致病机制以及防治方法等进行了综述,并对水稻寄生线虫致病机理的研究以及抗性品种、生物防治和诱导化合物的应用进行了探讨。     

Genetic and Phenotypic Diversity in the Graminaceous Cyst Nematode Complex, Inferred from PCR-RFLP of Ribosomal DNA and Morphometric Analysis

Graminaceous cyst nematodes form a group of eleven valid species including Heterodera avenae, Heterodera filipjevi and Heterodera latipons and constitute major pests to cereals. They are widely spread in circum-mediterranean areas where they are presumed to cause yield losses on bread and durum wheat. The objective was to document the diversity of these cereal cyst nematodes, in particular samples from Mediterranean regions, in comparison to species which develop on cultivated or wild grasses (H. arenaria, H. hordecalis, H. mani) and on rice or sugarcane (H. sacchari). Studies involved PCR-RFLP of ITS and morphometrics of the juvenile and cyst characters. UPGMA analysis of molecular data showed that the isolates segregated in two main clusters which seem to represent different evolutionary lineages. The H. avenae sensu lato cluster (I) contained H. arenaria, H. avenae, H. filipjevi and H. mani. The second cluster (II) contained isolates of H. hordecalis and H. latipons. Within H. avenae sensu lato, H. filipjevi was separated from the other related species with significant bootstrap value. The differentiation of H. arenaria, recognized for the first time based on molecular data, and H. mani with few restriction enzymes were the least significant. Intraspecific polymorphism allowed differentiation of isolates originating from Australia within H. avenae sensu stricto. The group H. hordecalis–H. latipons showed the greatest genetic variability between and within isolates. Two representatives of Heterodera sacchari, taxonomically included in the schachtiigrave group, were genetically as distant to this group as to the other graminaceous species belonging to either H. avenae sensu lato or H. hordecalis–H. latipons group. Results inferred from multivariate analysis applied on morphometrics of the cysts and juveniles showed agreement between genetic and phenotypic classifications. This study demonstrates the utility of combined molecular and classical methods to enhance our knowledge about the diversity within the complex of graminaceous cyst nematodes and to establish robust techniques to identify a wider set of nematode species.     

Effects of root diffusates of some weeds associated with corn plantations on the hatchability of the corn cyst nematode,Heterodera zeae

Twenty two summer weeds belonging to fifteen plant families and twelve winter weeds from eight plant families were tested to compare the effects of their root diffusates with the corn root diffusates (CRD) of cv. Giza 2 on the hatchability of the corn cyst nematode (CCN),Heterodera zeae.All tested weed root diffusates (WRD) successfully stimulated the hatchability of the cysts, but less than CRD did except the root diffusates (RD) ofRumex dentatus which failed to stimulate the nematode cysts.     

Host-suitability of black medick (<Emphasis Type="Italic">Medicago lupulina</Emphasis> L.) and additional molecular markers for identification of the pea cyst nematode <Emphasis Type="Italic">Heterodera goettingiana</Emphasis>

A survey was conducted in 16 fields cultivated with broad bean (Vicia faba L.) and garden pea (Pisum sativum L.) in nine localities of Apulia, southern Italy, to determine whether annual weeds were susceptible to the pea cyst nematode (PEACN), Heterodera goettingiana, and could therefore serve as alternate host for the nematode. The results of this study showed that black medick (Medicago lupulina L.) is a good host for the nematode increasing its population levels in the soil in the absence of the primary hosts. The identity of the PEACN was confirmed by integrative taxonomic approaches (classical, and molecular), resulting identical in all cases (broad bean and garden pea, as well as the spontaneous black medick infections). The phylogenetic analyses using ITS and coxI gene regions strongly support the identification of the populations of H. goettingiana from Italy. Also, ITS and coxI gene sequences were obtained from the same cyst, confirming the species identity in comparison to other nematodes and populations in the Goettingiana group, demonstrating that ITS and coxI gene regions of the PEACN are suitable molecular markers for accurate and unequivocal identification of the PEACN. Reproduction and histopathological analyses demonstrated a good host-suitability of black medick to the PEACN. This record enlarges the relatively narrow host-range of the pea cyst nematode and indicates the need to control M. lupulina to avoid the increase of the nematode population in the absence of the main host crop.     

Identification of the Beet Cyst Nematode Heterodera schachtii by PCR

PCR-RFLPs of ITS-rDNA and PCR with species-specific primers were developed for identification of cysts and juveniles of the beet cyst nematode Heterodera schachtii. Restrictions of PCR product by MvaI or ScrFI distinguish H. schachtii, H. betae, H. trifolii and H. medicaginis. RFLP profiles with eight restriction enzymes for these four nematode species are presented. Based on Internal Transcribed Spacer sequences of populations from several Schachtii group species, a specific primer for H. schachtii was designed, permitting amplification of the target sequence from juveniles and cysts of the beet cyst nematode. A duplex PCR protocol tested with a wide range of nematode samples is described.     

Involvement of OsNPR1/NH1 in rice basal resistance to blast fungus Magnaporthe oryzae

Rice blast disease, caused by the fungus Magnaporthe oryzae, is a major threat to worldwide rice production. Plant basal resistance is activated by virulent pathogens in susceptible host plants. OsNPR1/NH1, a rice homolog of NPR1 that is the key regulator of systemic acquired resistance in Arabidopsis thaliana, was shown to be involved in the resistance of rice to bacterial blight disease caused by Xanthomonas oryzae pv. oryzae and benzothiadiazole (BTH)-induced blast resistance. However, the role of OsNPR1/NH1 in rice basal resistance to blast fungus M. oryzae remains uncertain. In this study, the OsNPR1 gene was isolated and identified from rice cultivar Gui99. Transgenic Gui99 rice plants harbouring OsNPR1-RNAi were generated, and the OsNPR1-RNAi plants were significantly more susceptible to M. oryzae infection. Northern hybridization analysis showed that the expression of pathogenesis-related (PR) genes, such as PR-1a, PBZ1, CHI, GLU, and PAL, was significantly suppressed in the OsNPR1-RNAi plants. Consistently, overexpression of OsNPR1 in rice cultivars Gui99 and TP309 conferred significantly enhanced resistance to M. oryzae and increased expression of the above-mentioned PR genes. These results revealed that OsNPR1 is involved in rice basal resistance to the blast pathogen M. oryzae, thus providing new insights into the role of OsNPR1 in rice disease resistance.     

Estimation of Loss in Wheat Yield Due to the Cerealcyst Nematode Heterodera avenae

Abstract

The cereal cyst nematode, Heterodera avenae Wollenweber, 1924, occurs worldwide including India where it causes ‘molya’ disease of wheat and barley. Wheat yield losses due to the nematode were estimated during 1974-76 at a few villages in Mohindergarh district, Haryana, India, by applying dichloropropane-dichloropropene mixture and dibromochloropropane singly or in combination as soil f umigants. In light soil the cereal cyst nematode infestation at 4/5 eggs and larvae/g soil approximated the economic threshold level. However, depending on the initial population density of H. avenae and other field conditions, such as soil type, moisture, soil condition, chemical application method, chemical application time etc., the increase in yield of wheat cv. Kalyan Sona ranged from 8 to 49% at nematode infestation levels of about 6 to 13 eggs and larvae/g soil when treated with these nematicides.     

防治大豆孢囊线虫的生防放线菌初步筛选

大豆孢囊线虫是我国大豆主产区的主要病害之一,已造成严重产量损失,而且防治困难,目前人们试图利用生物防治来控制线虫病害,以达到农业可持续发展的目的。本试验采用稀释分离法,从东北和华北等地区采集土样分离获得400株放线菌,通过筛选获得6株对大豆孢囊线虫有强烈抑制作用的菌株,分别是C49、C44、C25-3、C58、H-2和H-4。其中C49和C44菌株的菌悬液对大豆孢囊线虫J2有较强的杀死作用,C25-3、H-2、C49、H-4和C44菌株的发酵液代谢物对大豆孢囊线虫孢囊孵化和J2活性均有不同程度的抑制作用。C49菌悬液处理48h后大豆孢囊线虫J2的校正死亡率达到77.78%;C25-3发酵液4×稀释浓度下,处理24 h后J2校正死亡率达到94.84%;C49、C25-3、C58对孢囊的孵化相对抑制率分别达89.50%、89.50%、88.80%,均与对照差异显著。     

Diversity and fertility of Fusarium sacchari from wild rice (Oryza australiensis) in Northern Australia, and pathogenicity tests with wild rice, rice, sorghum and maize

Research was undertaken to identify Fusarium spp. associated with an endemic wild rice (Oryza australiensis) community in northern Australia and determine the incidence of species reported as pathogens of tropical grain crops. A total of 92 isolates representing six Fusarium species were recovered from O. australiensis. Taxa were delimited based on morphological and phylogenetic (TEF-1α sequence) characters and identified as F. incarnatum-equiseti species complex (55 %), Gibberella fujikuroi species complex (GFSC, 27 %), F. longipes (14 %) and an unidentified Fusarium sp. (3 %). F. sacchari was dominant in the GFSC, a reported mycotoxin (beauvericin, fumonisin) producer and pathogen of sugarcane and sorghum. No isolates were identified as F. fujikuroi, the cause of Bakanae disease of rice in south-east Asia and other rice growing regions. Morphological, phylogenetic and phenetic (AFLP) analyses were in accordance, differentiating all F. sacchari isolates from other Fusarium taxa. The ratio of F. sacchari mating types (15 MAT-1:9 MAT-2) in the O. australiensis population was not significantly different from that expected under random mating (1:1). The effective population number (Ne) based on mating type (Ne(mt)) was 94 % of the count (total population). In contrast, that based on female-fertile isolates (Ne(f)) was 15 %, implying low frequencies of sexual reproduction among F. sacchari isolates. Pathogenicities of three F. sacchari genotypes were assessed against O. australiensis and commercial cultivars of rice, sorghum and maize. All F. sacchari isolates significantly (P?<?0.001) reduced emergence of O. australiensis and rice, but not sorghum or maize. Isolation frequencies of all genotypes from sorghum and maize and one isolate from O. australiensis were significantly (P?<?0.001) greater than the controls. There were no significant differences in F. sacchari re-isolation among rice treatments. Thus, F. sacchari is presumed to be a pre-emergent ‘damping off’ pathogen, endophyte or weak parasite of O. australiensis and rice and a root and crown pathogen of sorghum and maize.     

不同土壤质地对禾谷孢囊线虫侵染及种群动态的影响

为明确不同土壤质地对禾谷孢囊线虫Heterodera avenae发生危害的影响,分别测试了6种土壤质地中禾谷孢囊线虫的侵染、发育及其种群动态。结果显示：在整个小麦生长季,不同土质中禾谷孢囊线虫2龄幼虫（J2）的种群变化趋势一致。冬前土壤中少量线虫孵化,11月下旬各种土质中J2种群密度达到10.8~14.2条/100 mL土壤;4月初为J2发生高峰期,其种群密度为54.6~77.1条/100 mL土壤,且土壤中含壤土比例越高J2数量越大;J2在壤土与砂土比为6：1和1：0土壤中侵入根系的数量最多,单株根系J2数量为672.7~685.0条,且土壤中砂土比例越大J2侵入数量越少,相应地根系内3龄幼虫和形成的孢囊数量也越少;不同土质中不同时期孢囊内虫口减退率不同,但在生长季末不同土质中孢囊内虫口总减退率无显著差异。研究表明,土质对孢囊内虫口减退率无影响,土质沙性越大越不利于禾谷孢囊线虫的发生危害。     

Integrative diagnosis of carrot cyst nematode (<Emphasis Type="Italic">Heterodera carotae</Emphasis>) using morphology and several molecular markers for an accurate identification

Cyst nematodes obtained from commercial carrot fields in Ontario (Canada) and northern and southern Italy were subjected to morphological and molecular examination. Morphology of cyst cone tops, males and second-stage juveniles (J2) indicated the nematode species was the Carrot Cyst Nematode (CaCN), Heterodera carotae. The sequence of the Internal Transcribed Spacer (ITS), D2-D3 region of the 28S gene of ribosomal RNA, cytochrome oxidase I of mitochondrial DNA (coxI), and a heat shock protein gene (hsp90), from single cysts were also examined. Sequences of ITS and D2-D3 placed all the nematodes with Heterodera carotae and other Heterodera spp. belonging to the Goettingiana group in the same clade. The novel nine coxI sequences obtained also clustered in a well-supported phylogenetic clade for H. carotae. Similarly, the six new hsp90 sequences of H. carotae generated in this study were placed in a well-supported clade (PP = 1.00) together with other two sequences of H. carotae from Greece. Restriction Fragment Length Polymorphism (RFLP) of ITS-PCR products gave a restriction pattern for RsaI different than H. carotae but the other 6 restriction patterns were similar as described in former research. A diagnostic conventional PCR method was developed based on a primer set to be specific for H. carotae using coxI sequence. These primers were also used in real time PCR to generate a melt curve specific to H. carotae. Limit of detection for CaCN in conventional PCR reaction was a single J2.     

PCR zum halbquantitativen Nachweis von Rübenzystennematoden

Increased crop yield of nematode resistant sugar beet varieties compared to non-resistant varieties is dependent upon density of the sugar beet nematode, Heterodera schachtii, being below a certain threshold level. Therefore there is a requirement for an inexpensive quantitative laboratory test for H. schachtii. Currently the standard method to determine nematode densities relies on extraction of cysts from soil samples and their subsequent enumeration which is time-consuming. The method described in this article involves the extraction of nematodes obtained by a technique inducing the hatching of juveniles. Hatching of larvae is induced by incubation of the soil sample with “Acetox” followed by a Baermann funnel extraction. Instead of a visual estimation of nematode densities using a microscope, H. schachtii larvae are identified by a molecular diagnostic. The aim of this study is to distinguish between the three threshold infestation levels by comparing the intensities of PCR products derived from samples containing known numbers of target H. schachtii.     

旱稻孢囊线虫（Heterodera elachista）孵化特性研究

为了解旱稻孢囊线虫的孵化特性,从水稻根系及根系附近土壤分离孢囊,在室内离体条件下研究不同温度及水稻根系分泌物、土壤浸液、水稻根汁等因子对2龄幼虫孵化的影响,并观察不同温度下2龄幼虫的存活能力.结果表明:旱稻孢囊线虫孵化的适宜温度范围为28～32℃,且在该温度范围初孵2龄幼虫的存活率高、存活时间相对较长,35℃下孵化率及初孵2龄幼虫的存活率、存活时间均明显下降,40℃下无线虫孵化,20℃下孢囊线虫孵化率仅为0.9％,4℃下可延长2龄幼虫的存活时间.水稻分泌物、水稻土壤浸液和水稻20倍根汁对旱稻孢囊线虫孵化具有刺激作用,5倍根汁和4 mmol/L氯化锌溶液对线虫的孵化有抑制作用.上述结果可为制定旱稻孢囊线虫防治措施提供依据.     

Simple and rapid detection of rice false smut pathogen Ustilaginoidea virens in rice seeds

A method for simple and rapid detection of Ustilaginoidea virens in rice seeds was developed based on specific polymerase chain reaction (PCR). To design the specific primers for detection of U. virens, the comparison was made on 5.8S rDNA intra- and inter-specific variations in nucleotide sequences of U. virens and other pathogens: Fusarium verticillioides, Sclerotinia sclerotiorum, Tilletia barclayana, Fusarium graminearum and Magnaporthe oryzae. A 346-bp fragment could be amplified by using the specific primers uvr-F and uvr-R in U. virens, but not in other test fungi, indicating that the designed primers were specific for the detection of U. virens. U. virens was detected in nine of the 24 tested rice samples, indicating that 37.5% of the rice samples were contaminated with the false smut pathogen. The development of the simple and rapid detection technique using specific primers will be applicable for direct identification of U. virens in rice seeds to screen large numbers of samples.     

Nematodes of food legumes in the Mediterranean Basin1

Several endoparasitic nematodes have been reported on leguminous plants in the Mediterranean area. The most widespread are the root-lesion nematodes Pratylenchus mediterraneus, P. penetrans and P. thornei. Symptoms induced by these nematodes usually are not very impressive, but 50% yield loss of chickpea may occur. Among root-knot nematodes, Meloidogyne artiellia is associated with severe yield losses of chickpea in Italy, Spain and especially Syria. Tolerance limits of 0.14 and 0.02 of this nematode per ml soil are reported for winter and spring-sown chickpea, respectively. Meloidogyne incognita and M. javanica can be noxious to French bean and cowpea in sandy soil. The cyst nematode Heterodera goettingiana reduces yields of pea, broad bean and vetch when its population densities exceed 0.5, 1, and 2.1 eggs per g of soil, respectively. Heterodera ciceri occurs in northern Syria and Turkey and is responsible for economic yield losses of chickpea and lentil in fields infested with more than 1 or 2.5 eggs per g of soil, respectively. Pea and grass pea also suffer from infestation of this nematode. The stem and bulb nematode Ditylenchus dipsaci causes severe decline of broad bean, pea and probably lentil during wet seasons. Other nematodes, although present in moderate numbers, appear to have little importance.     

The growth of nematode 'tolerant' and 'intolerant' soyabeans as affected by phosphorus, Glomus intraradices and light

The effects of the vesicular-arbuscular mycorrhizal (VAM) fungus, Glomus intraradices , and the soyabean cyst nematode (SCN), Heterodera glycines , were studied singly and in combination on two soyabean cultivars, cv. Bragg (nematode intolerant) and cv. Wright (moderately nematode tolerant) grown in the greenhouse in soils with low (35 μg/g) and high (70 μg/g) phosphorus (P). Cultivar Wright grew better than cv. Bragg, showing a greater response to P and VAM, and was damaged less by SCN. These differences were not apparent, or were reversed, when the same cultivars were grown in growth chambers where lighting was sub-optimal. Cultivar Wright had a larger shoot:root ratio than cv. Bragg. This finding and the observed growth responses indicate that cv. Wright has a more efficient root system than cv. Bragg. This, we suggest, is the basis of both the greater VAM response observed in. and the greater nematode tolerance ascribed to, cv. Wright compared to cv. Bragg.     

Cereal diseases caused by <Emphasis Type="Italic">Fusarium graminearum</Emphasis>: from biology of the pathogen to oxidative burst-related host defense responses

Small grain cereals, such as wheat, barley and oats are considered among the most important food sources. Plant-parasitic nematodes play a considerable role in decreasing cereal yields. The three-major species of cereal cyst nematodes (CCN) Heterodera avenae, H. latipons, and H. filipjevi are distributed worldwide and cause considerable damage. This review provides information regarding the global distribution of these nematode species, yield loss due to CCN, their biology and pathogenic relation to plants, identification and control through agricultural practices, biological agents and resistance breeding. As morphological identification of CCN is difficult and time-consuming, several molecular techniques for the identification of these CCN species have been developed in recent years. The restrictions on the use of nematicides demand for resistance to CCN. Resistance genes in several lines are known and are used in numerous breeding programmes against CCN; pyramiding these resistance genes into high yielding cultivars that could become commercially available for farmers is progressing.     

Effect of fumonisin B<Subscript>1</Subscript> on the emergence,growth and ceramide synthase gene expression of cowpea (<Emphasis Type="Italic">Vigna unguiculata</Emphasis> (L.) Walp)

Rice production is currently expanding from the south-eastern regions of Australia into northern Australia where indigenous species of wild rice occur widely. A survey of fungal diseases on wild (Oryza australiensis, Oryza spp.) and cultivated rice (Oryza sativa) in North Queensland, Australia, in May 2014 revealed a diverse range of fungal genera species, including important pathogens of cultivated rice. Whilst a single isolate of Magnaporthe oryzae (causal agent of rice blast) was obtained from wild rice, Bipolaris oryzae (causal agent of brown spot) was the predominant pathogen detected under North Queensland conditions. For the first time for Australia, we report Rhizoctonia oryzae-sativae (causal agent of aggregate sheath spot) occurring on wild rice. Other pathogens detected on wild rice included Curvularia lunata, Cochliobolus intermedius, Cochliobolus geniculatus, and Fusarium equiseti present in the majority of wild rice samples. Nearby cultivated rice fields harboured additional pathogens not found in wild rice including Fusarium graminearum, Leptosphaeria spegazzinii and Cochliobolus lunatus, causing scab disease, glume blight and leaf blight, respectively. We also confirmed that Bipolaris oryzae from wild rice can infect cultivated rice. This study highlights the importance of wild rice species as alternative hosts harbouring pathogens of cultivated rice and the likely disease threats to expansion of cultivated rice into the same region(s) where wild rice is endemic.