Sugarcane grassy shoot disease

• SCGS disease is caused by a phytoplasma (Candidatus phytoplasma), which is one of the destructive pathogens of sugarcane (Saccharum officinarum L). In India, SCGS phytoplasmas are spreading at an alarming rate, adversely affecting yield of the sugarcane crop.[1][2][3]
• Phytoplasmas, formerly called mycoplasma-like organisms (MLOs), are a large group of obligate, intracellular, cell wallless parasites classified within the class Mollicutes.[2][3]
• Phytoplasmas are associated with plant diseases and are known to cause more than 600 diseases in several hundred plant species, including gramineous weeds and cereals.[4][5][6][7][8][9][10] The symptoms shown by infected plants include: whitening or yellowing of the leaves, shortening of the internodes (leading to stunted growth), smaller leaves and excessive proliferation of shoots, resulting in a broom phenotype and loss of apical dominance.[8]

Sugarcane is a vegetatively propagated crop, so the pathogen is transmitted via seed material and by phloem-feeding leafhopper vectors.[11][12] Saccharosydne saccharivora,[13] Matsumuratettix hiroglyphicus,[14] Deltocephalus vulgaris[15] and Yamatotettix flavovittatus[16] have been confirmed as vectors for phytoplasma transmission in sugarcane. Unconfirmed reports also suggest a spread through the steel blades (machetes) used for sugarcane harvesting..

Grassy appearance of phytoplasma-infected sugarcane plant

Phytoplasma-infected sugarcane plants show a proliferation of tillers, which give it typical grassy appearance, hence the name grassy shoot disease. The leaves of infected plants do not produce chlorophyll, and therefore appear white or creamy yellow. The leaf veins turn white first as the phytoplasma resides in leaf phloem tissue. Symptoms at the early stage of the plant life cycle include leaf chlorosis, mainly at the central leaf whorl. Infected plants do not have the capacity to produce food in the absence of chlorophyll, which results in no cane formation. These symptoms can be seen prominently in the stubble crop. The eye or lateral buds sprout before the normal time on growing cane. A survey of various fields of western Maharashtra showed grassy shoot with chlorotic or creamy white leaves was the most prevalent phenotype in sugarcane plants infected with SCGS.

Sugarcane crop showing iron deficiency

Interveinal chlorosis due to iron deficiency

Symptoms of iron deficiency (interveinal chlorosis) are very similar to those of SCGS. It shows creamy leaves, but no chlorosis occurs in leaf veins, and they remain green. In the case of severe iron deficiency, veins may lose chlorophyll in the absence of iron and appear similar to SCGS disease.[17][18] Iron deficiency is caused by a lack of iron nutrients in the soil; therefore, one may observe several plants showing symptoms of iron deficiency in localized patches in a field. Phytoplasma-infected plants, though, may occur anywhere in the field in a more random distribution. Treatment with 0.1% ferrous sulfate, either by spraying or supplying it through fertilizer cures iron deficiency, but phytoplasma-infected sugarcane does not respond to any treatment. Phytoplasma-infected plants growing in vitro show sensitivity to tetracycline.[17][18]

Phytoplasma-infected sugarcane can be recognized by visual symptoms, but there are limitations.[1]

• Visual symptoms occur only after considerable growth, normally two to three weeks after planting.[1]
• If not observed keenly, confusion may occur on differences between symptoms of SCGS disease and iron deficiency.
• In addition to above points, the poor relationship between symptoms and phytoplasma presence has been confirmed by earlier findings that symptoms alone are not reliable indicators of infection or identity. This highlights the importance of employing tests, such as the molecular tests, to verify associations between phytoplasma and putative disease symptoms. Also it suggests the inability to recognize a symptomless sugarcane harboring a phytoplasma could result in inadvertent exposure of sugarcane to a potential disease source.[19]
• Precise diagnosis is therefore necessary for effective disease identification and control. Though reliable, DNA hybridization,[20] electron microscopy[12] and PCR[21] techniques require specialized equipment and trained human resources. Among these, PCR is an accurate, economical and convenient method, which allows analysis of samples in a short time.

Detection of SCGS disease using phytoplasma rDNA PCR

In recent years, regions of the rRNA operon of the prokaryotic and eukaryotic organisms have been sequenced and are being used to develop PCR-based detection assays. These sequences are highly specific to the infecting organism.

Phytoplasma rRNA operon[21]

• The ribosomal DNA contains one transcriptional unit with a cluster of genes coding for the 18S, 5.8S and 28S rRNAs and two internal transcribed spacer regions, ITS1 and ITS2[22] in eukaryotes, and for 16S, 5S and 23S in prokaryotes . Previous studies have demonstrated the complex ITS regions are useful in measuring close genealogical relationships, because they exhibit greater interspecies differences than the smaller and larger subunits of rRNA genes.[10]
• The use of specific probes as selective PCR primers offers an impressive approach for the rapid identification of a large number of phytoplasma isolates.[1][10][21][23][24][25]
• The agarose gel electrophoresis image beside shows amplification of partial, 1.2kb long, 16S rDNA] of Candidatus phytoplasma, where total genomic DNA from a suspected sugarcane plant has been used as template DNA.[1]

A 96-well microtiter plate being used for ELISA

ELISA has been used as a diagnostic tool in medicine and plant pathology. Serological detection using a specific antiserum is an economical and convenient method that allows for both analysis of many samples in a short time and immunohistological observation of infected tissues.

• A phytoplasma-specific antibody can be prepared using the following steps:

1. Cloning of the phytoplasma gene fragment into an Escherichia coli expression vector;
2. Expression in E. coli and purification of the protein; and
3. Preparation of antibodies in rabbits.

• This technique has been used to develop antibodies to the immunodominant membrane protein of several phytoplasmas. These antibodies represent important tools in the detection, localization, and purification of phytoplasmas and the target antigen.[4][5][6][7][26][27][28]