208 1. Introduction Black root rot of cucurbitaceous crops causes severe root destruction leading to growth depression, non-vas- cular wilt and premature death of plants. The disease was originally described in gherkin (Cucumis sativus) roots in the Netherlands by Van Kesteren, who classified its causal agent as Phomopsis sclerotioides Kesteren (Van Kesteren, 1966). It has since spread to other European regions, including U.K., Germany, Denmark, Norway, France (Ebben and Last, 1973), and Italy (Cappelli et al., 2004). In Japan, the disease was first reported in 1985 in squash root which was the rootstock of a cucumber (Cucumis sativa) (Hashimoto and Yoshino, 1985). Since then, the disease has also been found in melon (Cucu- mis melo), watermelon (Citrullus lanatus), pumpkin (Cucurbita maxima), and other cucurbits, threatening the production of these major cucurbit crops with severe economic damage. Shishido et al. (2006) identified the causal agent of this disease as P. sclerotioides Kesteren through both morphological and phylogenetical analyses. Because of recent development in DNA sequence com- parisons, asexual state of genus, Phomopsis, has con- nected to sexual state, Diaporthe, which has priority over Phomopsis, and should be the generic name adopted for these taxa (Udayanga et al., 2012; Gomes et al., 2013), and thus I use Diaporthe sclerotioides as the name of the pathogen of this disease in this article. To control black root rot of cucurbits, solarization with a combination of soil fumigants such as chloropicrin has been widely applied in greenhouses, especially in warmer climate regions (Kobayashi et al., 1997). However, the disease has been consistently spreading in cucumber pro- duction areas in northern parts of Japan, where climate conditions are not warm enough to apply such tempera- ture-dependent measures. In addition, the disease has not yet been contained in the southern parts of Japan even though the incidence is sporadic and inconsistent (Shishi- do, 2006). The present paper offers a concise review of recent developments in knowledge about black root of cu- curbit crops as well as control measures against this dis- ease in Japan. 2. Taxonomy of Diaporthe sclerotioides Morphology The telemorphic stage of D. sclerotioides has not yet been discovered, while anomorphic stages of Diaporthe species are often characterized with specific pycinidia. D. sclerotioides forms pycinidium which are mostly sub- globose or spherical under the epidermis (Van Kesteren, 1966). In our experiment, pycnidia were found submersed in sterilized beanpods after three weeks of incubation, mostly globose and varying in size (200-500 μm in diam- eter) (Fig. 1A). The internal cavity of a pycnidium was often divided by protrusions of the proliferous layer (Fig. Black root rot caused by Diaporthe sclerotioides threatens cucurbit cultivation in Japan M. Shishido Graduate School of Horticulture, Chiba University, 648 Matsudo, Matsudo-shi, 271-8510 Japan. Key words: Black root rot, cucurbitaceae, Diaporthe sclerotioides, Abstract: In Japan, since black root rot of cucurbitaceous crops was found more than 30 years ago, the disease has caused severe economic losses to the cucurbit crop industry. Subsequent to the pathogen being correctly identified as Diaporthe sclerotioides based on morphology and DNA sequence, knowledge about DNA sequences has developed making techni- cal tools to detect and quantify the pathogen in natural samples of plants and soils available. In addition to chemical soil disinfectants, solarization and biological soil disinfestation have been developed as environment-friendly methods to effectively control this disease. Although it is difficult to apply such temperature-dependent methods in open-fields especially under cool climate conditions, an alternative approach, which changes soil pH to weak alkaline with amending steel converter slag, has also proved effective against the disease. In this mini-review, the process of D. sclerotioides iden- tification, detection and quantification methods developed for this fungus, host specificity, and disease control measures available and practiced in Japan are discussed concisely. Adv. Hort. Sci., 2014 28(4): 208-213 (1) Corresponding author: shishido@faculty.chiba-u.jp Received for publication 17 September 2014 Accepted for publication 5 November 2014 Mini review 209 1B). Numerous pycnospores (Fig. 1C), mostly ellipsoidal to ovoid, hyaline, 7-12 × 3-6 μm in size, usually with two guttules, were observed in the pycnidium (Fig. 1D) and apparently were produced on conidiogenous cells (Fig. 1E). Conidiophores filiform, hyaline, septate at the base, rarely branched, up to 25 μm, were formed from the inner cells of the locular walls. These only α-type conidiospores were unlike other Diaporthe species and no β-type were observed. Although we found that an isolate produced pycnidia and conidiospores, the event was very rare and no such organs have been reported in the natural environ- ment. Therefore, we suspect that conidiospores are not the primary source of inoculum of this pathogen. On the other hand, D. sclerotioides easily forms dull, grayish-brown mycelial mats on common agar media in- cluding potato dextrose agar (Shishido et al., 2006). The mycelium consisted of thin hyaline hyphae (2-5 μm in di- ameter) and thick hyaline to dark brown hyphae (10-20 μm in diameter) (Fig. 1F). A layer of the thick hyphae and dark brown, thick-walled cells formed a small sclerotium (Fig. 1G), and later became superficial or submersed pseu- do-sclerotia, mostly flattened, of various sizes (Fig. 1H). Because these pseudo-sclerotia are commonly observed on diseased roots as well, they are likely the primary in- ocula of black root rot of cucurbit crops. Molecular phylogeny Phylogenic analyses of the genus Diaporthe have been conducted in a number of studies (Rehner and Uechker, 1994; Zhang et al., 1998; Kanematsu et al., 2000; Farr et al., 2002; Murali et al., 2006; Shishido et al., 2006; Santos et al., 2010; Udayanga et al., 2012; Gomes et al., 2013). DNA sequences of the ITS regions of D. sclerotioides iso- lated from various parts of Japan formed a single distinct clade without differing from the ex-type D. sclerotioides (CBS 296.67) by even a single nucleotide (Shishido et al., 2006). The close kin species of D. sclerotioides were D. columnaris (Farr et al., 2002) and D. strumella var. longis- pora (Udayanga et al., 2012). These species differ 11 and 26 bases of the DNA sequences in their ITS regions out of 329 bases in total (ITS 1 and 2). The relationships detected with the DNA sequences of the ITS region were also found in other loci including elongation factor 1-α (Udayanga et al., 2012) and a mating type gene, MAT1-1-1 (Santos et al., 2010). Interestingly, we found no host-specific DNA sequenc- es within the ITS regions among the Japanese isolates of D. sclerotioides that originated from four different host spe- cies: melon, watermelon grafted on bottlegourd, pumpkin, and cucumber. Rehner and Uecker (1994) argued that the host-based species concept was not reliable for Diaporthe because of the genetic diversity among isolates of this ge- nus from various hosts. Kanematsu et al. (2000) supported this hypothesis by demonstrating that the phenotypic di- vergence of Phomopsis species, i.e. W type (mainly white colonies, weakly virulent, bearing both α- and β-type co- nidia) and G type (mainly gray colonies, highly virulent, bearing only α-type conidia) was dependent on their ITS sequences rather than on the host species. Since the Japa- nese D. sclerotioides isolates of black root rot are all G type, it may not be surprising that these isolates create a single clade in the phylogenetic tree. 3. Methods for detection and quantification of Dia- porthe sclerotioides Plant pathogenic fungi are usually detected by their growth on selective media or by biochemical, chemical, and immunological analyses. However, none of these con- ventional techniques are available for D. sclerotioides. Moreover, morphological identification of these fungi on nonselective media is time-consuming and requires expert knowledge of classical taxonomy. In recent years, PCR- Fig. 1 - Typical features of Diaporthe sclerotioides grown under gnotobi- otic conditions. A, Pycnidia on a sterilized beanpod. B, Vertical section of pycnidium. C, Numerous pycnospores mechanically released from pycnidium. D, Pycnospores. E, Pycnophores on the internal wall of pycnidium. F, Thick-walled, crenate aerial mycelium commonly found in various agar cultures. G, Masses of dark-colored mycelium, later become pseudomicrosclerotia. H, Small, brownish-black pseudomicrosclerotia on a boiled cu- cumber leaf. Reprinted from reference Shishido et al. (2006) with permission from the publisher. 210 based molecular techniques have contributed to the detec- tion and identification of various types of plant pathogens. Real-time PCR techniques combine the sensitivity of con- ventional PCR with the generation of a specific fluores- cent signal. This signal can be measured throughout the procedure, providing real-time analysis of the reaction ki- netics, and thereby allowing for quantification of specific DNA targets (Schena et al., 2004). Shishido et al. (2010) constructed conventional and real-time PCR primers to detect D. sclerotioides, utilizing a DNA sequence in the ITS region specific to this fungus. The designed primers, CPs-1 (forward) and CPs-2 (re- verse), successfully detected the fungus in soil and plant samples collected from fields naturally infested with the disease. Furthermore, the CPs-1/CPs-2 primer pair was applied to a real-time PCR assay with SYBR Green I, and the protocol achieved a linear response with a high corre- lation coefficient between input DNA and cycle threshold. However, because SYBR Green I is a DNA-intercalating dye without sequence specificity (Wittwer et al., 1997), and because DNA in natural samples usually contains un- known sequences, the amount of DNA will only be accu- rate if no presence of other DNA similar to this fungus is confirmed in the sample. This problem was solved using a TaqMan probe-based real-time PCR assay, which is highly specific, sensitive and quantitative (Shishido et al., 2013). In addition, the TaqMan probe-based protocol allows mul- tiplex real-time PCR, and thus using internal standard DNA such as GFP for soil samples (Klerks et al., 2004) and COX for plant samples (Weller et al., 2000), quanti- fication of the fungal DNA should be more accurate than the mono-plex counterparts. Table 1 summarizes primer sequences that can be used for detecting and quantifying D. sclerotioides DNA in natural samples. 4. Host range and specificity of Diaporthe sclerotioides In general, it is important in breeding programs to know if there are specific interactions between pathogenic microorganisms and host species. Although D. sclerotioi- des can cause black root rot only in cucurbit species, until recently little has been known about the degrees of either the host susceptibility to this disease or host specificity of the pathogen. Shishido et al. (2014) hypothesized that D. sclerotioides isolates were more infective and virulent to the cucurbit species from which the pathogens were origi- nally isolated than to other host species. They conducted cross-inoculation experiments using cucumbers, melons, pumpkins, watermelons, and bottlegourd (Lagenaria siceraria var. gourda), by inoculating 12 D. sclerotioides isolates from these cucurbit species. The virulence of the isolates was evaluated as the area under the disease prog- ress curve (AUDPC). All cucurbit species were suscepti- ble to each isolate, but AUDPCs were significantly differ- ent among the hosts as melon > cucumber ≥ watermelon ≥ bottlegourd ≥ pumpkin. The infectiveness of isolates, on the other hand, was assessed as the quantity of D. sclerotioides DNA detected in the hypocotyls of seedlings two weeks after inoculation using the TaqMan-based real-time PCR protocol described above. The fungal DNA quantities varied among the spe- cies in the same order as the AUDPCs. Orthogonal con- trasts indicated no specificity in either the fungal virulence or infectiveness between D. sclerotioides isolates and the cucurbit hosts from which these isolates originated (Fig. 2). Based on these results, the original hypothesis was re- futed and they concluded that though host susceptibility to black root rot varies among cucurbit species, D. sclero- tioides isolates are unlikely to have specificity to the host Table 1 - PCR protocols for detecting and quantifying Diaporthe sclerotioides in natural samples Detection of D. sclerotioides (Shishido et al., 2010) CPs-1 (forward) CPs-2 (reverse) 5´-GCCTCGGCGCAGGCCGGCCTCACC-3´ 5´-GGGGCCTTCCAGAACGAAATATAATTT-3´ Note: Not recommended for real-time PCR, Expected amplicon size: 392 bp Detection and quantification of D. sclerotioides (Shishido et al., 2013) CPs2f (forward) CPs2r1 (reverse) CPs2t (probe) 5´-ACTGCTTGGTGTTGGGGCACC-3´ 5´-TCCAGAACGAAATATAATTTACTACGCT-3´ 5´- [FAM]-AAAGGGCGGGCCCTGAAATCTAGTGGCGA-[TAMRA] -3´ Note: Applicable with SYBR Green I instead of TaqMan probe, Expected amplicon size: 101 bp Internal standard of soil samples (Klerks et al., 2004) FPGFP (forward) RPGFP (reverse) PYYGFP (probe) 5´-TGGCCCTGTCCTTTTACCAG-3´ 5´-TTTTCGTTGGGATCTTTCGAA-3´ 5´- [VIC]-AACCATTACCTGTCCACACAATCTGCCC-[TAMRA] -3´ Note: Applicable with SYBR Green I instead of TaqMan probe as an external standard. Internal standard of plant samples (Weller et al., 2000) COX-F (forward) COX-R (reverse) COX-P (probe) 5´-CGTCGCATTCCAGATTATCCA-3´ 5´-CAACTACGGATATATAAGAGCCAAAACTG-3´ 5´- [VIC]-AGGGCATTCCATCCAGCGTAAGCA-[TAMRA] -3´ Note: Applicable with SYBR Green I instead of TaqMan probe as an external standard. 211 species in terms of either virulence or infectiveness. This research implies that in practice seedlings and soils from infested areas should be handled carefully because the pathogen may spread to various cucurbit species irrespec- tive of the original host. Interestingly, the relationship between the AUDPC and D. sclerotioides DNA quantity in hypocotyls provided evi- dence that the virulence of this pathogen was not highly correlated with its infectiveness, although some degree of fungal invasion was obviously required for disease de- velopment (Fig. 3). The correlation analyses also showed that, although for most of the cucurbit species there were statistically significant correlations between AUDPCs and D. sclerotioides DNA quantities in the hypocotyls, their relatively low coefficients of determination indicated lim- ited associations for these variables, therefore suggesting that the virulence of D. sclerotioides may be due to factors in addition to infectiveness. 5. Control measures against black root rot of cucurbit crops Van Kesteren (1966) indicated that Cucurbita ficifolia was tolerant to the disease despite apparent root infection by D. sclerotioides. However, no true resistant variety or R-genes as such has been reported to black root rot. It is possible to use C. ficifolia for rootstocks of cucumber, and in fact it used to be a common practice in cucumber production in Japan. Unfortunately, the rootstock of C. ficifolia causes a fine white powder on the surface of the cucumber fruits, called “bloom”, primarily composed of silica (Mitani et al., 2011). In the late 1980s, cucumber without any bloom (bloomless cucumber) became popular in Japan because of its more attractive and distinctly shiny appearance; and thus C. ficifolia has no longer be used as rootstock of cucumber in the commercial production. Because D. sclerotioides is a soil-borne pathogen, soil disinfestation is a common control measure against black root rot of cucurbits. Although chloropicrin appears to be the most effective among disinfectant chemicals, its high toxicity to humans and unpleasant odor limits its popu- larity in practical applications (Shishido and Takeuchi, Fig. 2 - The area under the disease progress curve (AUDPC) for black root rot in cucumber, melon, pumpkin, watermelon, and bot- tlegourd plants (a), and the quantity of Diaporthe sclerotioides DNA detected in the hypocotyls of these plants (b) after root inoculation with 12 isolates of D. sclerotioides. In both charts, orthogonal contrasts were tested between original host-fungal isolate combinations and non-original host-fungal isolate com- binations with P values showing type I error probabilities of null hypotheses. The same letters indicate no significant differ- ence between plant species as determined by Tukey’s HSD test (P < 0.05). Error bars denote the standard error of the mean. Reprinted from reference Shishido et al. (2014) with permis- sion from the publisher. Fig. 3 - Correlations between the area under the disease progress curve (AUDPC) and the quantity of Diaporthe sclerotioides DNA de- tected in the hypocotyls of all the cucurbit species combined (a), cucumber (b), melon (c), pumpkin (d), watermelon (e), and bottlegourd (f) after root inoculation with 12 isolates of D. sclerotioides. Reprinted from reference Shishido et al. (2014) with permission from the publisher. 212 2005). Soil solarization is an alternative and environ- mentally-healthy measure against the disease since the pathogen is known to be heat-sensitive (Kobayashi et al., 1997). One of the problems in soil solarization is to main- tain high temperatures (37.5°C for 2 days or 35.0°C for 6 days) throughout the root range of soil depth, ca. 30 cm. In recent years, biological soil disinfestation (BSD), or anaerobic soil disinfestation (ASD) has been successfully applied to control some soil-borne pathogens and pests including Fusarium oxysporum, Ralstonia solanacearum, and parasitic nematodes (Momma et al., 2006; Lamers et al., 2010). BSD increases the effect of solarization by amending organic substances such as wheat bran or green manure crops under anaerobic conditions. Yokoyama et al. (2012) demonstrated that BSD with a low concentration of ethanol (0.5%-1.0%) was sufficiently effective to con- trol black root rot of cucumber. They recommend a low concentration of ethanol instead of wheat bran for the soil amendment because the former produces almost the same level of disease control efficacy to the latter with little un- pleasant odor during the anaerobic process. Although BSD may be a promising control measure against black root rot, the practice may only be applicable in protected facilities such as greenhouses and walk-in tun- nels in relatively warm climate regions. Therefore, other control measures are needed that are applicable in vast, un- protected fields especially in cool climate regions. Iwadate (2012) demonstrated that changing soil pH to weak alka- line, i.e. pH=7.5, with amending steel converter slag, sig- nificantly reduced the disease severity of cucumber in un- protected fields. All of these cucumber fields were in Iwate Prefecture, located in the north-eastern part of Japan, an area known for its cool climate. Although the mechanisms of disease suppression of this method have not yet been elucidated, it is certainly interesting since steel converter slag is easily available at a low cost as a byproduct of steel manufacturing. To date, only a few studies of biological control have been conducted regarding black root rot, e.g. Gliocladium roseum by Moody and Gindrat (1977) and Pseudomonas sp. by Fuchs and Defago (1991). Nonetheless, because D. sclerotioides is a slow growing fungus compared with oth- er major soil-borne fungal pathogens such as Fusarium, Rhizoctonia, and Pythium, the fungus may be less com- petitive in searching for nutrients and habitats in soil. In- terestingly, hypovirulent elements such as double-stranded RNA are also known to infect Diaporthe species (Ghab- rial, 2013). Although the detailed mechanism has not been elucidated, some isolates of D. sclerotioides significantly reduce in virulence (Shishido et al., 2014). Therefore, bio- logical control including hypoviruses may have potential for controlling black root rot of cucurbit crops in future. 6. Conclusions In Japan, more than 30 years have passed since black root rot was first reported in cucumber. Since then, the fun- gal isolates were correctly identified as Diaporthe sclero- tioides based on their morphology and DNA sequence. In addition, the knowledge of DNA sequence has developed technical tools for detecting and quantifying the pathogen in natural samples of plants and soils. On the other hand, aside from chemical soil disinfestation, environment- friendly control measures have also been developed by ap- plying solarization as well as biological soil disinfestation. Although such temperature-dependent methods may not properly be applicable to out-fields especially in cool cli- mate regions, an alternative measure by changing soil pH to weak alkaline with amending steel converter slag has proved effective to control this disease. Nonetheless, the area of its infestation is still expanding to northern parts of Japan. 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