Multi-gene analysis and morphology reveal novel Ilyonectria species associated with black foot disease of grapevines

Black foot is an important disease of grapevines, which has in recent years been recorded with increased incidence and severity throughout the world, affecting grapevines both in nurseries and young vineyards. In the past the disease has been associated with infections by Ilyonectria macrodidyma, Ilyonectria liriodendri, Campylocarpon fasciculare, and Campylocarpon pseudofasciculare. Based on published data, a high level of genetic diversity was detected among isolates of I. macrodidyma. To resolve this issue, we employed a multigene analysis strategy (based on the β-tubulin, histone H3, translation elongation factor 1-α, and the internal transcribed spacers on both sides of the 5.8S nuclear ribosomal RNA gene) along with morphological characterisation to study a collection of 81 I. macrodidyma-like isolates from grapevine and other hosts. Morphological characters (particularly conidial size) and molecular data (highest resolution achieved with histone H3 nucleotide sequence) enabled the distinction of six monophyletic species within the I. macrodidyma complex, four of which (Ilyonectria alcacerensis, Ilyonectria estremocensis, Ilyonectria novozelandica, and Ilyonectria torresensis) are described here. This work forms part of an effort by the International Council on Grapevine Trunk Diseases to resolve the species associated with black foot disease, which we believe will clarify their taxonomy, and therefore help researchers to devise control strategies to reduce the devastating impact of this disease.     

Truncatella angustata associated with grapevine trunk disease in northern Iran

In recent years, grapevine trunk diseases have gained growing attentions due to worldwide incidence of the disease. In a survey on fungal agents associated with grapevine trunk diseases in northern Iran, wood samples were collected from grapevines having the symptoms of declination. Isolation was made using routine plant pathology methods. A coelomycetous fungus with appendage-bearing conidia was recovered from symptomatic tissues. Based on morphological and cultural characteristics, the causal agent of the disease was identified as Truncatella angustata. The identity of the species was further confirmed by sequence data of internal transcribed spacer-rDNA region. A phylogeny inferred using sequence data obtained in this study, together with the sequences from GenBank, clustered our isolates together with T. angustata known from other host plant species. Pathogenicity tests performed on detached shoots of grapevines led to the same symptoms as observed in field conditions. This is first study on the pathogenicity of T. angustata on grapevine in Iran and first report on the occurrence of T. angustata on grapevine in Iran.     

Colonization of barley roots by endophytic fungi and their reduction of take-all caused by Gaeumannomyces graminis var. tritici

Fungal root endophytes obtained from natural vegetation were tested for antifungal activity in dual culture tests against the root pathogen Gaeumannomyces graminis var. tritici. Fifteen isolates, including Acremonium blochii, Acremonium furcatum, Aspergillus fumigatus, Cylindrocarpon sp., Cylindrocarpon destructans, Dactylaria sp., Fusarium equiseti, Phoma herbarum, Phoma leveillei, and a sterile mycelium, selected based on the dual culture test, were inoculated on barley roots in growth tubes under axenic conditions, both in the absence and presence of G. graminis var. tritici. All isolates colonized the rhizosphere and very often the root cortex without causing disease symptoms and without affecting plant growth. Eight isolates significantly reduced the symptoms caused by G. graminis var. tritici, and 6 of them reduced its presence in the roots.     

Uneven Distribution of Mating‐Type Alleles Among Togninia minima Isolates,One of the Causal Agents of Leaf Stripe Disease on Grapevines in Northwest Iran

Togninia minima is the main fungal species associated with grapevine leaf stripe disease worldwide. This species is mainly known from its asexual state in nature; nevertheless, a biallelic heterothallic mating strategy has been confirmed for this species based on in vitro crossing studies. There are no data available on the incidence of an active sexual cycle within the populations of this species in many grapevine‐producing countries as well as Iran. The possibility of a clandestine sexual cycle within the Iranian isolates of T. minima was evaluated by analysing the distribution and frequency of the mating‐type alleles on a microspatial and a macrogeographical scales. Towards this aim, a total of 90 T. minima isolates were recovered from grapevines with esca disease from the vineyards in north and north‐western Iran. A multiplex PCR method previously designed by authors was applied for simultaneous identification and determination of the mating‐type alleles in T. minima populations. The results on the screening of mating‐type alleles using multiplex PCR method revealed the mating‐type identity of 77 isolates as Mat1‐2 and 23 isolates as Mat1‐1. Our results showed that both Mat1‐1 and Mat1‐2 isolates are present in a single vineyard and even on single vines. The distribution of mating‐type alleles in the sampled area skewed from the 1 : 1 ratio (77 : 23); however, co‐occurrence of both mating types in a single vineyard and even on single vines is suggestive for the presence of an active sexual cycle for T. minima in north‐western Iran.     

Uwe Joachim Nebert ‐ 70 Jahre

Esca is a very important and destructive disease of grapevine in many grapevine producing countries. Several groups of fungi have been reported from grapevines with esca disease symptoms; however, Phaeoacremonium species are the main hyphomycete fungi involved in this disease. In recent years, esca disease symptoms with consequent decline disease have frequently been observed in vineyards of north-western Iran. Nevertheless, the involvement of Phaeoacremonium spp. with esca disease symptoms in this region remains unknown. During 2008–2010 growing seasons, wood samples were collected from vines showing typical esca disease symptoms such as interveinal leaf chlorosis with subsequent necrosis and various types of internal wood deterioration in north-western parts of Iran. A total of 44 Phaeoacremonium-like hyphomycetes were recovered from sampled materials. Fungal isolates were subjected to tentative morphological identification and were further characterised by using sequenced data from ITS-rDNA and β-tublin gene. Phylogeny inferred using sequence data from ITS-rDNA region and β-tublin gene revealed that Phaeoacremonium aleophilum and P. mortoniae together with Sarocladium strictum (syn. Acremonium strictum) are associated with esca disease symptoms in this region. The pathogenicity of Phaeoacremonium spp. and Sarocladium strictum were verified by the inoculation of cutting root and stems under greenhouse conditions. Isolates of P. aleophilum and P. mortoniae were the most virulent based on the length of vascular necrosis on woody stems that followed by Sarocladium strictum. This is first study on the pathogenicity of Sarocladium strictum on grapevine worldwide and the first report on the occurrence of P. mortoniae on grapevines in north-western Iran.     

Botryosphaeria dothidea and Other Fungi Associated with Excoriose and Dieback of Grapevines in Portugal

In a study of fungi associated with symptoms of excoriose in Portugal, the most prevalent ones were found to be Botryosphaeria dothidea and Phomopsis viticola. In artificial inoculations, these two fungi infected grapevines causing dark lesions on the shoots and bud mortality. They were re-isolated from the shoot lesions and necrotic buds. Two further species of Botryosphaeria , namely B. obtusa and B. stevensii , were also isolated and they caused small lesions on wounded shoots but did not infect unwounded shoots. B. dothidea was also isolated from grapevines with a dieback of the trunk. These isolates were more virulent on young shoots and one-year-old canes than the ones from grapevines with excoriose.     

DNA phylogeny, morphology and pathogenicity of Botryosphaeria species on grapevines

Several species of Botr yosphaeria are known to occur on grapevines, causing a wide range of disorders including bud mortality, dieback, brown wood streaking and bunch rot. In this study the 11 Botryosphaeria spp. associated with grapevines growing in various parts of the world, but primarily in South Africa, are distinguished based on morphology, DNA sequences (ITS-1, 5.8S, ITS-2 and EF1-α) and pathological data. Botryosphaeria australis, B. lutea, B. obtusa, B. parva, B. rhodina and a Diplodia sp. are confirmed from grapevines in South Africa, while Diplodia porosum, Fusicoccum viticlavatum and F. vitifusiforme are described as new. Although isolates of B. dothidea and B. stevensii are confirmed from grapevines in Portugal, neither of these species occurred in South Africa, nor were any isolates of B. ribis confirmed from grapevines. All grapevine isolates from Portugal, formerly presumed to be B. ribis, are identified as B. parva based on their EF1-α equence data. From artificial inoculations on grapevine shoots, we conclude that B. australis, B. parva, B. ribis and B. stevensii are more virulent than the other species studied. The Diplodia sp. collected from grapevine canes is morphologically similar but phylogenetically distinct from D. sarmentorum. Diplodia sarmentorum is confirmed as anamorph of Otthia spiraeae, the type species of the genus Otthia (Botryosphaeriaceae). A culture identified as O. spiraeae clustered within Botryosphaeria and thus is regarded as probable synonym. These findings confirm earlier suggestions that the generic concept of Botryosphaeria should be expanded to include genera with septate ascospores and Diplodia anamorphs.     

Genetic and pathogenic diversity of Neofusicoccum parvum in New Zealand vineyards

Genetic diversity of 50 isolates of Neofusicoccum parvum, the predominant species of the Botryosphaeriaceae recovered from grapevines displaying symptoms of dieback and decline in New Zealand, was compared to that of isolates from Australia, South Africa, and California. The eight universally primed polymerase chain reaction (UP-PCR) primers distinguished 56 genotypes, with only four clonal pairs found. Seven main groups were identified in a neighbour-joining (NJ) tree with isolates from different regions and vineyards of New Zealand, Australia, and California distributed in different groups, indicating a high level of intra and intervineyard genetic variation. All of the South African isolates were positioned in a separate UP-PCR group, indicating that these isolates were less related to the other N. parvum isolates. When compared to fungi that reproduce sexually the genetic diversity and Shannon diversity indices were low (0.076-0.249; 0.109-0.367, respectively), genetic identity levels were high (0.76-0.95), and genetic distance levels were low (0.04-0.27). The large number of genotypes and the low number of clones in the New Zealand N. parvum populations may be explained by parasexual recombination as anastomosis was observed between nonself pairings. Pathogenicity tests using isolates from different UP-PCR groups inoculated onto either green shoots or 1-y-old grapevines detected virulence diversity, indicating intra and intervineyard variation between isolates, however, no correlation was detected between UP-PCR group and virulence.     

Isozyme Analysis and Soluble Mycelial Protein Pattern in Iranian Isolates of Several formae speciales of Fusarium oxysporum

A total of 13 representative isolates of Fusarium oxysporum f. sp. melonis (FOM) from Iran, USA and France, eight isolates of seven formae speciales from Iran and one isolate of F. oxysporum f. sp. niveum from the USA were compared based on isozyme analysis and soluble mycelial protein pattern. Isozyme analyses of alkaline phosphatase (ALP), catalase (CAT), esterase (EST), malate dehydrogenase (MDH), superoxide dismutase (SOD) and xanthine dehydrogenase (XDH) revealed polymorphism among the F. oxysporum isolates in which 22 electrophoretic phenotypes (EP) were determined. At least 10 putative loci for these six enzymes were detected and they were all polymorphic. Maximum genetic diversity was observed in CAT, EST and XDH loci. Using UPGMA, the 22 isolates were separated into three main groups with one of the groups divided into two subgroups. Group I included isolates belonging to five formae speciales from Iran, whereas group II that included FOM isolates from both Iran and the USA was divided into two subgroups each containing the vast majority of the respective isolates from either country. Group III constituted FOM isolates from France and one pathogenic isolate on pepper from Iran. FOM isolates representing five different geographical regions from Iran belonged to two different races of 1 and 1,2Y and one vegetative compatibility group (VCG)0134 and thus were genetically homologous. Isozyme polymorphism in these isolates was highly correlated with VCG and geographical origins and to a lesser extent with races. Variations in soluble protein profile in FOM isolates were correlated with genetic distances determined in isozyme analysis. This study suggests that isozyme analysis could be a useful tool for identifying genetic diversity not only in FOM but also several formae speciales of F. oxysporum.     

Characterization of Closterovirus-like Particles Associated with Grapevine Leafroll Disease

Abstract A purification procedure (Z ee et al. 1987) for closterovirus-like particles associated with grapevine leafroll disease was improved after various sources of plant tissues, extraction buffers, pH values, and centrifugation techniques had been examined. An antiserum against the CA-4 isolate was produced and used in ELISA. The virus-like particles of the NY-1 isolate measured in crude plant preparations were about 1,800 to 1,900 nm long. The molecular weight of coat protein of the NY-1and NY-2 isolates were ca. 43 × 10³ daltons in SDS-PAGE analysis; and bands having this molecular weight reacted in Western blotting tests with specific polyclonal and monoclonal antibodies. A large dsRNA molecule (ca. 10 × 10⁶ Mr) along, with lower molecular weight species were isolated from leafroll diseased grapevines, but not from healthy grapevines. Seven leafroll isolates were tested for their serological relatedness in a protein A-gold labelling immunosorbent electron microscopy, assay. Results indicated that serologically distinct serotypes existed, and mixed infection of grapevines with different scrotypes was common. A grouping of isolates into serotype I, II, III, IV is proposed.     

Control of Pierce's Disease by Phage

Pierce’s Disease (PD) of grapevines, caused by Xylella fastidiosa subsp. fastidiosa (Xf), is a limiting factor in the cultivation of grapevines in the US. There are presently no effective control methods to prevent or treat PD. The therapeutic and prophylactic efficacy of a phage cocktail composed of four virulent (lytic) phages was evaluated for control of PD. Xf levels in grapevines were significantly reduced in therapeutically or prophylactically treated grapevines. PD symptoms ceased to progress one week post-therapeutic treatment and symptoms were not observed in prophylactically treated grapevines. Cocktail phage levels increased in grapevines in the presence of the host. No in planta phage-resistant Xf isolates were obtained. Moreover, Xf mutants selected for phage resistance in vitro did not cause PD symptoms. Our results indicate that phages have great potential for biocontrol of PD and other economically important diseases caused by Xylella.     

Fertility status and distribution of mating type alleles of the rice blast fungus, Magnaporthe grisea in northern Iran

This study was carried out using 155 monoconidial isolates collected from different areas of two major rice growing provinces in northern Iran, including 94 isolates from Guilan and 59 isolates from Mazandaran. Among 94 isolates from Guilan, 92 and two isolates recovered from rice and crabgrass (Digitaria sp.), respectively. All 61 rested isolates from Mazandaran were recovered from rice. All isolates were evaluated for in vitro sexual fertility and mating type status by pairing with Mat 1-1 and Mat 1-2 fertile standard hermaphrodite isolates including Br48 and Th12 (Mat 1-1) and KA9 and TH16 (Mat 1-2). Of 155 isolates, 98 (63.2%) were fertile and 57 (36.8%) were infertile and produced no perithecium when mated with standard isolates. Among 98 fertile isolates, 96 isolates were identified as Mat 1-1 and two isolates as Mat 1-2. All Mat 1-1 isolates were obtained from rice and two Mat 1-2 isolates obtained from crab grass. No Mat 1-2 isolate was identified from rice in this study. Both mating types were found in Guilan but all isolates recovered from Mazandaran were identified as Mat 1-1. Male fertility predominated in fertile Mat 1-1 and Mat 1-2 isolates from all sampling sites in northern Iran, and no female fertility was detected. This is the first report of existence of Mat 1-2 allele in Magnaporthe grisea population in Iran.     

Genetic Diversity of Echinococcus granulosus in Center of Iran

Hydatid cyst caused by Echinococcus granulosus is one of the most important parasitic diseases around the world and many countries in Asia, including Iran, are involved with this infection. This disease can cause high mortality in humans as well as economic losses in livestock. To date, several molecular methods have been used to determine the genetic diversity of E. granulosus. So far, identification of E. granulosus using real-time PCR fluorescence-based quantitative assays has not been studied worldwide, also in Iran. Therefore, the aim of this study was to investigate the genetic diversity of E. granulosus from center of Iran using real-time PCR method. A total of 71 hydatid cysts were collected from infected sheep, goat, and cattle slaughtered in Isfahan, Iran during 2013. DNA was extracted from protoscolices and/or germinal layers from each individual cyst and used as template to amplify the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) (420 bp). Five cattle isolates out of 71 isolates were sterile and excluded from further investigation. Overall, of 66 isolates, partial sequences of the cox1 gene of E. granulosus indicated the presence of genotypes G1 in 49 isolates (74.2%), G3 in 15 isolates (22.7%), and G6 in 2 isolates (3.0%) in infected intermediate hosts. Sixteen sequences of G1 genotype had microgenetic variants, and they were compared to the original sequence of cox1. However, isolates identified as G3 and G6 genotypes were completely consistent with original sequences. G1 genotype in livestock was the dominant genotype in Isfahan region, Iran.     

The influence of genotypic variation on metabolite diversity in populations of two endophytic fungal species

The relationship between metabolite production and genotypic diversity in two endophytic fungi was investigated. We selected populations of Cylindrocarpon destructans and Heliscus lugdunensis from the roots of a single tree. A total of 49 isolates of both species were selected and classified by simple genotypic tests (random amplified polymorphic DNA analysis and rDNA-ITS sequencing). In a blind test, the ability of these fungi to produce natural products was tested by ethyl acetate extraction of hyphae and culture filtrates, followed by high-performance liquid chromatography analysis (HPLC). A positive relationship was found between genotype classification and the pattern of natural products produced by a given isolate. To test the robustness of this correlation, a discriminate selection procedure was carried out by collecting fungal isolates from a second site and selecting a sub-set of the population, on the basis of genotypic variability. This sub-set of fungal isolates produced greater numbers of unique metabolites than those selected indiscriminately.     

Phylogenetic Analysis of Phaeosphaeria Species Using Mating Type Genes and Distribution of Mating Types in Iran

Phaeosphaeria species are pathogenic on wheat, barley and a wide range of wild grasses. To analyze mating type loci of the Phaeosphaeria species and investigate mating type distribution in Iran, we sequenced mating type loci of 273 Phaeosphaeria isolates including 67 isolates obtained from symptomatic leaves and ears of wheat, barley, and wild grasses from two wheat-growing region in Iran as well as 206 isolates from our collection from other regions in Iran which were isolated in our previous studies. Mating type genes phylogeny was successfully used to determine the species identity and relationships among isolates within the Phaeosphaeria spp. complex. In this study, we reported seven new host records for Phaeosphaeria species and the Phaeosphaeria avenaria f. sp. tritici 3 group was first reported from Iran in this study. Mating type distribution among Phaeosphaeria species was determined. Both mating types were present in all sampling regions from Iran. We observed skewed distribution of mating types in one region (Kohgiluyeh va Boyer-Ahmad) and equal distribution in the other region (Bushehr). However, when considering our entire dataset of 273 Iranian Phaeosphaeria isolates, the ratio of mating types was not deviated significantly from 1:1 suggesting possibilities for isolates of opposite mating type to interact and reproduce sexually, although the sexual cycle may infrequently occur in some regions especially when the climatic conditions are unfavorable for teleomorph development.     

The Occurrence and Pathogenicity of Cylindrocarpon, Cylindrocladium and Fusarium on Calluna vulgaris and Erica spp. in England and Scotland

Cylindrocarpon, Cylindrocladium and Fusarium spp. were often isolated from the woody roots, stem-base and lower foliage of diseased container-grown Calluna vulgaris and Erica spp. plants collected from English and Scottish nurseries. The highest isolation frequencies were obtained for Cylindrocarpon destructans, Cylindrocladium scoparium, Cylindrocladium ilicicola. Fusarium tricinctum, Fusarium avenaceum and Fusarium sporotrichioides . Isolations of these fungi were made more frequently from diseased plants that were at least 1-year-old. The percentage incidence of Cylindrocarpon, Cylindrocladium and Fusarium spp. did not differ between Scottish and English nurseries. Cylindrocarpon destructans. Cylindrocladium ilicicola and C. scoparium were more pathogenic to rooted C. vulgaris cuttings than F. tricinctum. F. avenaceum or F. sporotrichioides in laboratory and glasshouse tests. The high isolation frequency of Cylindrocarpon. Cylindrocladium and Fusarium spp., and their pathogenicity in tests, suggests that these fungi are involved in root, stem-base and lower foliage diseases in crops of C. vulgaris and Erica spp. The importance of these findings for the integrated control of diseases on ericaceous plant nurseries is discussed.     

Neonectria and Cylindrocarpon: the Nectria mammoidea group and species lacking microconidia

Neonectria (Hypocreales: Nectriaceae) species having Cylindrocarpon anamorphs that lack microconidia and chlamydospores include: Neo. discophora var. discophora, Neo. discophora var. rubi, stat nov. et comb. nov., Neo. lucida, comb. nov., Neo. viridispora, sp. nov. and Neo. westlandica, comb. nov. Perithecia of these species are red and perithecial anatomyis of the N. mammoidea type, with a palisade of hypha-like cells in the outer perithecial wall. These species occur on recently dead or dying trees. Perithecia of Neo. betulae, sp. nov and Neo. dumontii, sp. nov. are anatomically and biologically similar to those of Neo. discophora. The only known culture of Neo. betulae remained sterile, while Neo. dumontii has not been cultured; their anamorphs are presumed to be Cylindrocarpon. Analyses of mit ssu rDNA sequences indicate that Neonectria/Cylindrocarpon is monophyletic. Within the genus, species having N. mammoidea type perithecia are paraphyletic. Most species cluster with Neo. discophora, but Neo. westlandica and Neo. trachosa are basal to a clade that includes species that do not have a N. mammoidea-type perithecium. Nectria fuckeliana clusters independently of Neonectria and Nectria. Although reported to have a Cylindrocarpon anamorph, fresh ascospore isolates of N. fuckeliana did not produce Cylindrocarpon macroconidia but produced acremonium- or verticillium-like anamorphs. A key to nectriaceous species of Neonectria that have Cylindrocarpon anamorphs that lack microconidia and chlamydospores and/or that have a N. mammoidea type perithecial wall anatomy is presented. New combinations are proposed for other species formerly included in Nectria that have non-microconidial Cylindrocarpon anamorphs: Neonectria cinnamomea, Neo. jungneri, Neo. platycephala, Neo. phaeodisca and Neo. verrucospora.     

Biotypes of Agrobacterium tumefaciens in Hungary

Isolates of Agrobacterium tumefaciens from Hungary were separated into three biotypes on the basis of their physiological characters. Biotypes 1 and 2 corresponded with those of Keane et al . (1970). The most common isolates were of biotype 2. Isolates from grapevines formed a separate biotype which might be distinguished from biotype 1 by D-(–)tartrate and malonate utilization. Many isolates with biotype-intermediate characters were found. Isolates utilizing D-(–)tartrate, erythritol and malonate were included into biotype 2, although many of them were 3–ketolactose positive. Biotypes were not separated geographically and biotype 1 and 2 apparently occurred together.     

The homogeneity of vanB gene cluster among enterococcal isolates in Iran

Aims:  This study aimed to analyse the diversity of the vanB gene cluster in enterococcal species isolated from sewage treatment plants (STP) in Tehran, Iran.
Methods and Results:  The enterococcal isolates were collected from three sewage treatment plants in Tehran, Iran, during 2005. A total of 203 enterococcal isolates, collected over six rounds of sampling from three STPs were tested for the presence of vanB gene. Long-PCR showed that amongst the isolates, three Enterococcus faecium , one Enterococcus gallinarum and one Enterococcus casseliflavus harboured the complete vanB gene cluster.
Restriction fragment length polymorphism (RFLP) of the vanB1 gene cluster (5900 bp) from the isolates showed an identical pattern to a standard strain of Enterococcus faecalis (V583). None of the isolates were able to transfer the vanB gene in conjugation experiments. Different pulsed-field gel electrophoresis patterns were obtained for the three E. faecium isolates with vanB gene clusters .
Conclusions:  Our results indicated that the dissemination of vanB is not widespread in Tehran. Although only a few vanB positive isolates were detected, vanB was found in several enterococcal species.
Significance and Impact of the study:  In view of the lack of information on vanB resistance genes and their diversity in Iran, knowledge of the global dissemination of vanB genes in Enterococcus spp. is noteworthy.     

Morphological and molecular characterization of Diplodia seriata,the causal agent of canker and twig dieback disease on mulberry in Iran

Mulberries are cultivated for different purposes: for feeding larvae of silkworm, Bombyx mori, as fresh and dry food resources, in wood instrument industry, in pharmaceutical industry and as outdoor ornamental trees in Iran. In recent years, twig and branch canker disease symptoms have been noticed on mulberry trees in northwestern parts of Iran. Diplodia isolates were repeatedly recovered from symptomatic tissues. Based on cultural and morphological features, the isolates were identified as Diplodia seriata. The identity of the isolates was further confirmed using sequence data from internal transcribed spacer (ITS)-rDNA and ef-1α gene. A phylogenetic analysis using ITS sequence data clustered the isolates obtained in this study together with known Diplodia seriata isolates of other woody hosts from GenBank. Inoculation studies carried out on white mulberry twigs using an excised shoot method revealed that the isolates are pathogenic on this host. D. seriata have been reported from other woody host plant species such as Juglans nigra and Vitis vinifera in Iran, however, to the best of our knowledge, the occurrence of D. seriata on mulberry trees is new for Iran. The distribution and reaction of different Morus spp. to D. seriata remain to be studied.